Particle structures comprising sterols and saponins

ABSTRACT

The present invention pertains to complexes comprising sterols and saponins. The complexes are capable of binding a genetic determinant including a polynucleotide. The complexes may further comprise a lipophilic moiety, optionally a lipophilic moiety comprising a contacting group and/or a targeting ligand, and/or a saccharide moiety. The complexes may further comprise an immunogenic determinant and/or an antigenic determinant and/or a medicament and/or a diagnostic compound. The complexes may in even further embodiments be encapsulated by an encapsulation agent including a biodegradable microsphere. The present invention also pertains to pharmaceutical compositions and methods of treatment of an individual by therapy and/or surgery, methods of cosmetic treatment, and diagnostic methods practised on the human or animal body.

This application is a continuation of U.S. application Ser. No.10/114,957, filed on Apr. 4, 2002, now allowed, which claims the benefitof U.S. provisional application Ser. No. 60/308,609 filed on Jul. 31,2001; and of Danish application number PA 200100560, which was filed onApr. 4, 2001, all incorporated herein by reference in their entireties.All patent and nonpatent references cited in the application, or in thepresent application, are also hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to complexes of sterols and saponinglycosides. The complexes are capable of interacting with bioactiveagents including genetic determinants, such as e.g. polynucleotides. Thecomplexes pertaining to the present invention can be used to facilitatethe transport of polynucleotides, including DNA and derivatives thereof,across cellular membranes.

Acting as carriers of various bioactive agents and genetic determinants,the complexes provide a means for introducing e.g. a polynucleotide intoa patient, a predetermined region of the patient, or a predeterminedbiological cell of the patient, in order e.g. to express a genecomprised by the polynucleotide and/or to regulate the expression ofgenes being expressed in the biological cell in vivo and/or in vitro.

More particularly, the present invention relates to novel methods fortransfecting a biological cell, to complexes involved in such methods,and to diagnostic methods and therapeutic methods for treating a patientby e.g. gene therapy and DNA-vaccination.

BACKGROUND OF THE INVENTION

Several medical applications utilizing genetic determinants have evolvedin recent years. In such applications, the introduction of whole genesor specific nucleic acids into cells is of central importance. Thisprocess is often referred to as gene transfection independently of theorigin of the cells, the sequence and character of the nucleic acid, andirrespective of whether the transfer is performed in vivo or ex vivo.

To facilitate the process of gene transfection several differentapproaches have been developed. Such approaches include among others i)using biological vectors (including viral vectors), ii) associating anucleic acid with a cationic liposome, iii) associating a nucleic acidwith peptides covalently linked to a transfection agent, and iv) coatingminute gold particles by nucleic acids and using the coated particlesfor a bio-ballistic transfer.

The major iscom constituents are quillaja saponins and cholesterol. Theprocedure for preparation of iscoms comprises solubilization ofamphipathic polypeptides in preferably nonionic detergents, addition ofQuillaja saponins, cholesterol, and possibly also phosphatidylcholine.In the presence of amphipathic proteins, iscom particles are formed onremoval of the detergent.

Morein (see e.g. U.S. Pat. No. 4,578,269) was the first to describe thatiscoms not only formed a very characteristic structural complex, butalso possessed significant immunogenic properties when amphipathicantigens were inserted into this complex by hydrophobic interaction.Conventional iscoms (immunostimulating complexes) have since been usedfor vaccine formulations and combine a multimeric presentation of anantigen with an adjuvant functionality. (see e.g. U.S. Pat. No.6,080,725 to Marciani).

SUMMARY OF THE INVENTION

The present invention in one aspect relates to a complex comprising atleast one sterol and/or at least one saponin, wherein the at least onesterol or the at least one saponin is capable of forming anelectrostatic interaction or a hydrophobic interaction with at least onebioactive agent, including a genetic determinant, including apolynucleotide, including DNA and derivatives thereof. When the at leastone sterol and the at least one saponin is incapable of forming anelectrostatic interaction or a hydrophobic interaction with the at leastone bioactive agent as described herein above, the complex comprises atleast one contacting group capable of contacting the at least onebioactive agent, including a genetic determinant, including apolynucleotide, including DNA and derivatives thereof, by means of aninteraction selected from an electrostatic interaction and a hydrophobicinteraction and an interaction resulting from intercaation of thegenetic determinant by the contacting group.

According, in one aspect of the invention there is provided a complexcomprising

i) at least one first sterol and/or at least one second sterol,

wherein the at least one second sterol is capable of contacting agenetic determinant by means of an interaction selected from anelectrostatic interaction and a hydrophobic interaction, and

wherein the at least one first sterol and/or the at least one secondsterol is capable of forming a complex with at least one first saponinand/or at least one second saponin, and

ii) at least one first saponin and/or at least one second saponin,

wherein the at least one second saponin is capable of contacting agenetic determinant by means of an interaction selected from anelectrostatic interaction and a hydrophobic interaction, and

wherein the at least one first saponin and/or the at least one secondsaponin is capable of forming a complex with at least one first steroland/or at least one second sterol, and optionally

iii) at least one contacting group for contacting a genetic determinantby means of an interaction selected from an electrostatic interactionand a hydrophobic interaction,

with the proviso that the at least one contacting group is present whenno second sterol and no second saponin is present in the complex.

The complexes according to the invention are useful for bindingpolynucleotides including naturally occurring nucleic acids includingDNA and RNA, and derivatives thereof, including, but not limited topeptide nucleic acids (PNA) and locked nucleic acids (LNA). The boundpolynucleotide can subsequently be transferred into a biological cellincluding any animal cell or human cell.

The complexes according to the present invention may in one preferredembodiment adopt a micro-particle structure in the form of a cage-likematrix similar to that known as an immune stimulating complex (iscom).Beside iscom structures, the interaction between sterols and saponinshave been reported to result in a variety of different structuralentities, including entities such as e.g. lattices, honeycombes, rods,and amorphic particles, all of which structural entities are covered bythe present invention. However, other structures and matrix formationsare also envisaged by the present invention which is in no way limitedto iscom-like structures or matrixes.

In the case where the complexes according to the present invention doform iscoms, or iscom-like structures, such iscoms or structures may beprepared e.g. essentially as described in European patent EP 0 109 942B1.

Accordingly, a glycoside solution, containing fx cholesterol,phospholipid, and one or more glycosides (fx Quillaja components) withhydrophobic and hydrophilic domains in a concentration of at least acritical micelle-binding concentration, is formed and a complex isgenerated. The complex may subsequently be isolated and/or purified.

Optionally, as a first step, the component to be inserted into thecomplex, fx a bioactive agent, including a polynucleotide, such as DNAand derivatives thereof, an immunogenic agent, an antigenic agent, atherapeutic agent, a diagnostic agent, and the like, can be mixed withone or more solubilizing agents, whereby complexes are formed betweenthe component and solubilizing agents, after which the components areseparated from the solubilizing agent and e.g. transferred directly tothe glycoside solution.

In line with the present invention, the glycoside solution may initiallybe mixed with a polynucleotide. It is possible to proceed from a matrixthat can be made by solubilizing at least one sterol in a solutionagent, adding at least one glycoside or at least one saponin, andoptionally the other lipophilic moieties, after which addition thesolution agent may be removed, if it is proving unacceptable to thefinal product.

The matrix may be transferred to a water solution in which its separateparts are not soluble. The solubilizing agent can be removed through eggel filtration, ultra filtration, dialysis, or electrophores. The matrixcan then be purified from surplus of first sterol and saponin e.g. byultracentrifugation, through a density gradient, or through gelfiltration. The solubilizing agent may be any one of those mentioned inU.S. Pat. No. 5,679,354, which is incorporated herein by reference.

In one preferred embodiment, the complexes according to the inventionare formed essentially as described in Example 1 herein.

Accordingly, in one preferred embodiment, there is provided a method forpreparation of a complex comprising

i) at least one first sterol and/or at least one second sterol,

wherein the at least one second sterol is capable of contacting agenetic determinant by means of an interaction selected from anelectrostatic interaction and a hydrophobic interaction, and

wherein the at least one first sterol and/or the at least one secondsterol is capable of forming a complex with at least one first saponinand/or at least one second saponin, and

ii) at least one first saponin and/or at least one second saponin,

wherein the at least one second saponin is capable of contacting agenetic determinant by means of an interaction selected from anelectrostatic interaction and a hydrophobic interaction, and

wherein the at least one first saponin and/or the at least one secondsaponin is capable of forming a complex with at least one first steroland/or at least one second sterol, and optionally

iii) at least one contacting group for contacting a genetic determinantby means of an interaction selected from an electrostatic interactionand a hydrophobic interaction,

with the proviso that the at least one contacting group is present whenno second sterol and no second saponin is present in the complex, andfurther optionally

iv) at least one lipophilic moiety,

wherein said method comprises the steps of

a) mixing a sterol composition comprising at least one first steroland/or at least one second sterol, with

b) a saponin composition comprising at least one first saponin and/or atleast one second saponin, and

c) at least one lipophilic moiety, and

d) at least one organic solvent,

wherein the steps a) to d) may be carried out simultaneously, orsequentially, in any order, and optionally

e) removing surplus reactants and/or purifying the prepared complexes.

The organic solvent is preferably selected from ethanol, DMSO, and DMF,and the solvent is preferably present in an amount of at the most 25%(vol/vol), such as at the most 20% (vol/vol), for example at the most15% (vol/vol), such as at the most 10% (vol/vol), for example at themost 8% (vol/vol), such as at the most 6% (vol/vol), for example at themost 4% (vol/vol), such as at the most 2% (vol/vol), for example at themost 1% (vol/vol), such as at the most 0.5% (vol/vol), for example atthe most 0.1% (vol/vol).

First and Second Sterols and Saponins

The following general distinction is made between first and secondsterols. First and/or second sterols can be naturally occurring sterolssynthesised as secondary metabolites by many organisms. They may also besynthetic, or they may be made by chemical synthesis or enzymaticsynthesis either in vitro or in vivo. First sterols are generallyincapable of forming an association with a genetic determinant asdefined herein, whereas such an association is formed between secondsterols and the genetic determinant.

Similarly, first and/or second saponins can be any saponin as definedherein comprising as an aglycone part either i) a triterpene part, ii) asteroid part, or iii) a steroid alkaloid part. The saponins may benaturally occulting or synthetic, or they may be made by chemicalsynthesis or enzymatic synthesis either in vitro or in vivo. Firstsaponins are generally incapable of forming an association with agenetic determinant as defined herein, whereas such an association isformed between second saponins and the genetic determinant.

Consequently, any sterol will either be a first sterol, or a secondsterol, and any saponin will either be a first saponin, or a secondsaponin. In principle, first and/or second sterols, as well as firstand/or second saponins, may be either anionic, neutral, or cationic. Theterms cationic sterols and cationic saponins shall denote sterols andsaponins, respectively, carrying a net positive charge at pH 7.0. Secondsterols and second saponins are preferably cationic sterols and cationicsaponins, respectively, whereas first sterols and first saponins arepreferably anionic or neutral sterols and saponins, respectively.

Accordingly, the second sterols and/or second saponins preferablycomprise at least one positively charged moiety or reactive group atpH=7.0, and this positively charged moiety or reactive group isaccording to one preferred embodiment of the invention capable ofcontacting a bioactive agent, including a genetic determinant, by meansof an electrostatic interaction.

In another embodiment, the second sterols and/or second saponinspreferably comprise an uncharged moiety or non-polar reactive group, andthis uncharged moiety or non-polar reactive group is according toanother preferred embodiment of the invention capable of contacting abioactive group, including a genetic determinant, by means of ahydrophobic interaction.

A combination of electrostatic interactions and hydrophobic interactionscan also be used for generating an association between bioactive agentsand second sterols and/or saponins.

When the complexes according to the present invention comprise no secondsterol and no second saponin, the complexes comprise at least onecontacting group capable of contacting a genetic determinant by meanseither of an electrostatic interaction, or a hydrophobic interaction.

The contacting group is necessary in order for such complexes to formthe desired association with the genetic determinant. However, thecontacting group may also be present in complexes comprising a secondsterol and/or a second saponin. The teen “contacting group” as usedherein will also refer to any moiety of a second sterol and/or a secondsaponin capable of forming an association with a genetic determinant,including an association generated by an electrostatic interactionand/or a hydrophobic interaction.

Preferred contacting groups comprise at least one lipophilic moietycapable of forming an association with the complex according to theinvention made up by sterols (first and/or second) and/or saponins(first and/or second).

In addition to acting as a “docking group” or “anchor” for contactinggroups according to the invention, lipophilic moieties may also serve tofacilitate a saponin-sterol complex formation essentially withoutserving, during or after complex formation, as a “docking group” for acontacting group or any other functional group forming part of thesaponin-sterol complex. The purpose of using lipophilic moieties maythus be two-fold: The lipophilic moieties according to the invention mayact—during or after saponin-sterol complex formation, preferably duringsaponin-sterol complex formation—as a facilitator of complex formation,and they may, independently of this action, also act as a “dockinggroup” for any functional group including contacting groups forcontacting a genetic determinant and/or targeting ligands for targetingthe complexes according to the invention.

Lipophilic moieties such as phospholipids are preferably present duringthe process of forming the complexes according to the invention. Thepresence of e.g. phospholipids facilitates complex formation over abroad concentration range. Accordingly, phosphatidyl choline may beadded when mixing saponins and sterols during complex formation.

It is preferred to use a phospholipid with a positively chargedheadgroup such as phosphatidyl ethanolamine. One reason for this is theinclusion into the complex of an overall positive charge capable offacilitating or resulting in an interaction with negatively chargedpolynucleotides including ribonucleic acids. This interaction willfacilitate the uptake of the polynucleotides across a cellular membraneand into a biological cell for subsequent integration and/or translationand/or polypeptide expression. Phosphatidyl choline is an example ofanother phospholipid capable of being used in connection with thepresent invention.

Additional moieties of a contacting group may be either lipophilic orhydrophilic, depending on the nature of the association formed with thegenetic determinant. One preferred additional moiety of the contactinggroup is a ionic group, or a charged group, preferably a positivelycharged group, including a group comprising a positive charge at pH=7.0.Examples of such groups are illustrated for lipophilic moieties in FIGS.6, 7 and 8. Such lipophilic moieties can be obtained from, among others,Avanti Polar Lipids, Inc. Alabaster, Ala.

It is thus possible to imagine a series of different compositions of thecomplexes according to the invention. The invention in preferredembodiments relates to

i) complexes comprises at least one first sterol and at least one firstsaponin and at least one contacting group,

ii) complexes comprising at least one first sterol and at least onesecond saponin,

iii) complexes comprising at least one first sterol and at least onesecond saponin and at least one contacting group,

iv) complexes comprising at least one first sterol and at least onefirst saponin and at least one second saponin,

v) complexes comprising at least one first sterol and at least one firstsaponin and at least one second saponin and at least one contactinggroup,

vi) complexes comprising at least one second sterol and at least onefirst saponin,

vii) complexes comprising at least one second sterol and at least onefirst saponin and at least one contacting group,

viii) complexes comprising at least one second sterol and at least onesecond saponin,

ix) complexes comprising at least one second sterol and at least onesecond saponin and at least one contacting group,

x) complexes comprising at least one second sterol and at least onefirst saponin and at least one second saponin,

xi) complexes comprising at least one second sterol and at least onefirst saponin and at least one second saponin and at least onecontacting group,

xii) complexes comprising at least one first sterol and at least onesecond sterol and at least one first saponin,

xiii) complexes comprising at least one first sterol and at least onesecond sterol and at least one first saponin and at least one contactinggroup,

xiv) complexes comprising at least one first sterol and at least onesecond sterol and at least one second saponin,

xv) complexes comprising at least one first sterol and at least onesecond sterol and at least one second saponin and at least onecontacting group,

xvi) complexes comprising at least one first sterol and at least onesecond sterol and at least one first saponin and at least one secondsaponin,

xvii) complexes comprising at least one first sterol and at least onesecond sterol and at least one first saponin and at least one secondsaponin and at least one contacting group,

including any composition comprising any combination of the complexeslisted herein immediaterly above, i.e. any combination of complexes i)to xvii) Such combinations may be of value when attempting to direct ortarget different complexes to different regions of a patient.

The mechanism by which the contacting group interacts with nucleic aciddepends upon the character of the contacting group in question. Oneexample of a contacting group is a cationic (positively charged)derivative of a cholesterol which is capable of forming an interactionwith a genetic determinant by e.g. forming an electrostatic interactionwith the backbone of a polynucleotide including any DNA-backbone.Another example is a lipid-tailed acridin-compound that intercalateswith double stranded DNA.

In addition to sterols (first and/or second), saponins (first and/orsecond), and contacting groups comprising a lipophilic moiety, thecomplexes according to the present invention may further comprise atargeting ligand for targeting a particular polynucletide, including anucleic acid, to a specific cell surface or tissue. This may beaccomplished by incorporation of specific targeting ligands and/orreceptor binding molecules and/or ligands into the structure of thecomplexes according to the invention.

Accordingly, two different methods for the formation of the complexesaccording to the invention are provided, in one method the contactinggroup are incorporated during the formation of the particles, and inanother method the contacting group is added to preformed particles,optionally in the form of iscom-matrices.

One principal aspect of the invention is thus the provision of a novelsystem that enhances the uptake of polynucleotides including nucleicacids by combining—in one preferred embodiment—i) the ability ofiscom-structures to associate with, and penetrate into or throughmembranes containing cholesterol, with ii) the property of contacting orassociating with polynucleotides including nucleic acids. In particularfor the application of vaccination with naked DNA the complexesaccording to the invention is likely to reduce the required amount ofDNA.

It should be noted that the invention is not limited to complexescapable of forming an association with polynucleotides. The complexesaccording to the invention in other preferred embodiments are capable ofcontacting or forming an association with polypeptides and/orpolynucleotides.

Accordingly, the present invention in one embodiment pertains tocomplexes comprising components also forming a part of traditionaliscoms. However, the complexes according to the present invention aredifferent from conventional iscoms in a number of ways. Firstly, thecomplexes according to the present invention are capable of binding abioactive agent including a genetic determinant in the form of e.g. apolynucleotide. Secondly, the complexes in one preferred embodiment arecharacterised by a zeta-potential which is less negative—or morepositive—than about −50 mV.

Accordingly, there is provided a complex having a zeta-potential whichis less negative or more positive than about −50 mV, such as azeta-potential of about −45 mV, for example a zeta-potential of about−40 mV, such as a zeta-potential of about −37 mV, for example azeta-potential of about −35 mV, such as a zeta-potential of about −32mV, for example a zeta-potential of about −30 mV such as azeta-potential of about −29 mV, for example a zeta-potential of about−28 mV, such as a zeta-potential of about −27 mV, for example azeta-potential of about −26 mV, such as a zeta-potential of about −25mV, for example a zeta-potential of about −20 mV, such as azeta-potential of about −15 mV, for example a zeta-potential of about−10 mV, such as a zeta-potential of about −5 mV, for example azeta-potential of about 0 mV, such as a zeta-potential of about 5 mV,for example a zeta-potential of about 10 mV, such as a zeta-potential ofabout 15 mV, for example a zeta-potential of about 20 mV, and preferablya zeta-potential of less than 50 mV.

The complexes in preferred embodiments may thus have a zeta-potential offrom about −50 mV to about −40 mV, such as from about −40 mV to about−35 mV, for example of from about −35 mV to about −30 mV, such as fromabout −30 mV to about −25 mV, for example of from about −25 mV to about−20 mV, such as from about −20 mV to about −15 mV, for example of fromabout −15 mV to about −10 mV, such as from about −10 mV to about −5 mV,for example of from about −5 mV to about 0 mV, such as from about 0 mVto about 5 mV, for example of from about 5 mV to about 10 mV, such asfrom about 10 mV to about 15 mV, for example of from about 15 mV toabout 20 mV, such as from about 20 mV to about 30 mV, for example offrom about 30 mV to about 40 mV, such as from about 40 mV to about 50mV.

Using as a standard reference point a complex comprising i) any givensaponin(s) of interest and ii) cholesterol as a first sterol, complexesaccording to the invention comprising i) any given saponin(s) ofinterest, ii) cholesterol as a first sterol, and iii) any given secondsterol of interest, preferably a cholesterol derivative as listed inFIG. 5 herein, said complexes according to the invention have azeta-potential which is at least about 5 mV less negative or morepositive than said reference complexes: such as a zeta-potential whichis at least about 10 mV less negative or more positive than saidreference complexes, for example a zeta-potential which is at leastabout 15 mV less negative or more positive than said referencecomplexes, such as a zeta-potential which is at least about 20 mV lessnegative or more positive than said reference complexes, for example azeta-potential which is at least about 25 mV less negative or morepositive than said reference complexes, such as a zeta-potential whichis at least about 30 mV less negative or more positive than saidreference complexes, for example a zeta-potential which is at leastabout 35 mV less negative or more positive than said referencecomplexes, such as a zeta-potential which is at least about 40 mV lessnegative or more positive than said reference complexes, for example azeta-potential which is at least about 45 mV less negative or morepositive than said reference complexes, such as a zeta-potential whichis at least about 50 mV less negative or more positive than saidreference complexes.

The values of the zeta-potentials for the complexes of the presentinvention are determined in part by the inclusion of e.g. cationicsecond saponins and/or cationic second sterols into the complexesaccording to the present invention.

The molar ratio between saponins (first and second) and sterols (firstand second) in complexes according to the present invention ispreferably from less than 1000:1 to preferably more than 1:1000.Preferred ratios are about 100:1, for example about 80:1, such as about60:1, for example about 50:1, such as about 40:1, for example about30:1, such as about 25:1, for example about 20:1, such as about 18:1,for example about 16:1, such as about 14:1, for example about 12:1, suchas about 10:1, for example about 9:1, such as about 8:1, for exampleabout 7:1, such as about 6:1, for example about 5:1, such as about 4:1,for example about 3:1, such as about 2:1, for example about 1.9:1, suchas about 1.8:1, for example about 1.7:1, such as about 1.6:1, forexample about 1.5:1, such as about 1.4:1, for example about 1.3:1, suchas about 1.2:1, for example about 1.1:1, such as about 1:1, for exampleabout 1:1.1, such as about 1:1.2, for example about 1:1.3, such as about1:1.4, for example about 1:1.5, such as about 1:1.6, for example about1:1.7, such as about 1:1.8, for example about 1;1.9, such as about1:2.0, for example about 1:2.5, such as about 1:3, for example about1:3.5, for example about 1:4, such. as about 1:4.5, for example about1:5, for example about 1:5.5, such as about 1:6, for example about 1:7,such as about 1:10, for example about 1:20, such as about 1:40, forexample about 1:60, such as about 1:80, for example about 1:100.

Without being bound by theory, the complexes according to the presentinvention may according to one presently preferred hypothesis adopteither an iscom-like structure, or a structure that does not resemblesuch a structure when contacting or being associated with either apolynucleotide and/or a polypeptide comprising e.g. natural or syntheticamino acids and variants thereof. When being connected by the complexes,the polynucleotides and/or polypeptides are according to one preferredhypothesis in a degradation-resistant conformation, or in a conformationless prone to degradation, as compared to the conformation of thepolynucleotide or the polypeptide when it is not associated with thecomplex according to the invention The invention thus in one preferredembodiment relates to complexes comprising a polynucleotide and/or apolypeptide that is less likely to be degraded by nucleases andproteases, respectively, under practical circumstances.

Accordingly, there is provided in one preferred embodiment a method foradministration of a polynucleotide and/or a polypeptide to an individualin need thereof, said polynucleotide and/or said polypeptide beingadministered in association with the complexes according to theinvention in a conformation that is not susceptible to degradation underpractical circumstances—or less susceptible to degradation underpractical circumstances—as compared to the degradation taking place whenthe polynucleotide and/or the polypeptide is administered in the absenceof the complexes under substantially similar conditions, includingconditions wherein the polynucleotide and/or the polypeptide isadministered in combination with conventional carriers or adjuvants.

Consequently, the present invention in one particular embodiment relatesto complexes in the form of modified iscoms that are used as carriersfor bioactive agents including polynucleotides and other geneticdeterminants which are desirably transfected into a biological cell bymeans of an interaction of the complex with the cellular membrane.

Complexes according to the invention may thus comprise one or moreselected from the group consisting of bioactive agents, immunogenicdeterminants, genetic determinants, enzymes, adjuvants and medicaments.Hence, by way of example a complex according to the present inventionmay comprise both a genetic determinant and an antigenic determinant,such as a nucleic acid and a polypeptide.

The complexes according to the invention may further comprise—inaddition to a bioactive agent and/or a genetic determinant, preferably apolynucleotide, i) a lipophilic moiety, optionally a lipophilic moietycomprising a contacting group and/or a targeting ligand, and/or ii) asaccharide moiety, preferably, but not limited to a saccharide moietyforming part of a naturally occurring saponin. It is particularlypreferred that the complexes according to the invention comprise asaccharide moiety when it is intended to direct the complexes tocellular surfaces known to contain saccharide binding receptors.

The complexes according to the invention may thus further comprise abioactive agent and/or a genetic determinant and/or an immunogenicdeterminant and/or an antigenic determinant and/or a medicament and/or atherapeutic agent and/or a diagnostic agent.

The complexes may in even further embodiments be encapsulated by anencapsulation agent including a liposome and/or a biodegradablemicrosphere. By analogy to the complexes, the liposome and/or thebiodegradable microsphere may comprise a targeting ligand suitablyassociated with the microsphere for targeting the microsphere to aparticular location in a human or animal body. Preferred targetingligands include, but are not limited to, ligands having affinity toreceptors on antigen presenting cells, including e.g. dendritic cellsand macrophages. When used for therapeutic purposes, the ligands aretargeted to e.g. vitamin receptors, folate receptors, high affinity IL-2receptors, growth factor receptors, such as EGF-receptors, and the like.

First saponins and/or second saponins and/or first sterols and/or secondsterols may also be present in e.g. the biodegradable microsphere. It isparticularly preferred in one embodiment that the microsphere has anoverall positive charge, i.e. contains more positively charged groupsthan negatively charged groups.

The present invention also pertains to pharmaceutical compositions andmethods of treatment of an individual by therapy and/or surgery, methodsof cosmetic treatment, and diagnostic methods practised on the human oranimal body.

There is also provided a method for manufacturing the complexesaccording to the invention, and in even further aspects the presentinvention relates to using the complexes according to the invention, orcompositions comprising such complexes, including pharmaceuticalcompositions, in the manufacture of a medicament for treating acondition in an individual in need of such treatment.

In addition, the present invention relates to a kit-of-parts, whereinsaid kit-of-parts comprises a complex according to the invention and atleast one immunogenic determinant. The present invention also relates toa kit-of-parts, wherein said kit-of-parts comprises a complex accordingto the invention and at least one immunogenic determinant and at leastone genetic determinant. For example the immunogenic determinant maycomprise an antigenic determinant.

The kit-of-parts according to the invention—as well as the complexesthemselves—can be used in a method for raising a desirable immuneresponse in a patient. Said method comprise the steps of i) providing acomplex according to the invention, and ii) providing a geneticdeterminant such as a polynucleotide such as DNA and/or iii) providingan immunogenic determinant such as antigenic determinant such as apeptide such as an epitope, iv) mixing said complex with said geneticdeterminant and/or said immunogenic determinant, v) administering saidmixture to a patient in an amount effective to raise a desirable immuneresponse in said patient, and vi) raising said desirable immuneresponse.

In preferred embodiments, the method comprises the further step ofadministering, simultaneously or sequentially in any order, a) a complexcomprising a genetic determinant, and b) a complex comprising animmunogenic determinant such as an antigenic determinant. The geneticdeterminant in one embodiment encodes the immunogenic/antigenicdeterminant, or part thereof. The immunogenic determinant Is preferablya lipid associated peptide (lipo-peptide, e.g. a lipo-protein or a partthereof). When the above method comprises the steps of sequentiallyadministering a) a complex comprising a genetic determinant, and b) acomplex comprising an immunogenic determinant, it is thus possible toadminister initially a genetic determinant and then boost after asuitable time period any initial immune response by subsequentlyadministering a complex comprising an immunogenic determinant,preferably an immunogenic determinant comprising an epitope encoded bythe initially administered genetic determinant. Initial administrationof an immunogenic determinant followed by administration after asuitable time period of a genetic determinant. Likewise, it is possibleto administer to a patient—simultaneously or sequentially in anyorder—i) a complex comprising a genetic determinant, and ii) a peptideincluding an epitope administered according to any state of the artmethod, as well as administering to said patient—simultaneously orsequentially in any order—i) a complex comprising an immunogenicdeterminant, including a peptide epitope, and ii) a polynucleotide suchas DNA administered according to any state of the art method.

The above complexes, or the individual components thereof, as well asthe genetic and immunogenic determinants can be provided in individualvials ready for use in a kit-of-parts. In this way it is possible to mixcomplexes according to the invention with genetic determinants, such aspolynucleotides, such as DNA, and/or with immunogenic determinants suchas antigenic determinants, such as peptides, such as epitopes.

DEFINITIONS

Aglycone: Part of a saponin glycoside, linked to saccharide part througha glycosidic bond.

Amphiphilic moiety: Any moiety, including a lipid, comprising asynthetic, semi-synthetic (modified natural) or naturally-occurringcompound having a water-soluble, hydrophilic portion and awater-insoluble, hydrophobic portion. Preferred amphiphilic compoundsare characterized by a polar head group, for example, aphosphatidylcholine group, and one or more nonpolar, aliphatic chains,for example, palmitoyl groups.

Antigenic determinant: Any determinant or substance with the capabilityof binding an antibody, or a binding fragment thereof, and inducing aspecific antigen response. The antigenic determinant may comprise oressentially consist of an epitope that forms part of a polypeptide andelicits a specific antibody response when the whole polypeptide is usedas an antigen. Such epitopes are confined to a single or a few loci onthe molecule in question.

Bioactive agent: Any a substance which may be used in connection with anapplication that is therapeutic or diagnostic, such as, for example, inmethods for diagnosing the presence or absence of a disease in a patientand/or methods for the treatment of a disease in a patient. “Bioactiveagent” refers to substances, which are capable of exerting a biologicaleffect in vitro and/or in vivo. The bioactive agents may be neutral,positively or negatively charged. Suitable bioactive agents include, forexample, prodrugs, diagnostic agents, therapeutic agents, pharmaceuticalagents, drugs, oxygen delivery agents, blood substitutes, syntheticorganic molecules, proteins, peptides, vitamins, steroids, steroidanalogs and genetic determinants, including nucleosides, nucleotides andpolynucleotides.

Cationic lipid: Any lipid carrying a net positive charge at pH 7.0.

Cationic saponin: Any saponin carrying a net positive charge at pH 7.0.

Cationic sterol: Any sterol carrying a net positive charge at pH 7.0.

Contacting group: Group comprising a lipophilic moiety and a moietycapable of association with a genetic determinant by means of either i)electrostatic interaction, or ii) hydrophobic interaction.

Complex: Formation of saponins and sterols capable of interacting withgenetic determinants or immunogenic determinants. The interaction canresult from electrostatic interactions and/or hydrophobic interactionsformed between on the one hand i) a saponin and/or a sterol, and on theother hand ii) a genetic determinant and/or an immunogenic determinant.When this is the case, the, saponin and/or the sterol is termed a secondsaponin and/or a second sterol, respectively. Complexes devoid of secondsaponins and second sterols form electrostatic and/or hydrophobicinteractions with genetic determinants and/or immunogenic determinantsby means of a contacting group. When the contacting group forms part ofa saponin or a sterol, said saponin or sterol is by definition a secondsaponin or a second sterol. Contacting groups may be present in acomplex independently of the presence of second saponins and/or secondsterols. Accordingly, contacting groups can be present in a complex alsocomprising second saponins and/or second sterols without said contactinggroup forming part of said second saponins and/or second sterols. Theoverall charge of a second saponin or a second sterol can be neutral oreven anionic, as long as the contacting group of the saponin or sterolin question comprises at least one positively charged moiety at pH=7capable of forming an association with a genetic determinant. Contactinggroups can also be neutral in which case predominantly hydrophobicinteractions with a genetic determinant are formed.

Degenerated polynucleotide sequence: Polynucleotide encoding apolypeptide and comprising an altered sequence of nucleotides ascompared to a polynucleotide comprising a predetermined sequence ofnucleotides and encoding a predetermined polypeptide, wherein thepolypeptide and the predetermined polypeptide have the same biologicalactivity.

Diagnostic agent: Any agent which may be used in connection with methodsfor imaging an internal region of a patient and/or diagnosing thepresence or absence of a disease in a patient. Diagnostic agentsinclude, for example, contrast agents for use in connection withultrasound imaging, magnetic resonance imaging (MRI), nuclear magneticresonance (NMR), computed tomography (CT), electron spin resonance(ESR), nuclear medical imaging, optical imaging, elastography,radiofrequency (RF) and microwave laser. Diagnostic agents may alsoinclude any other agents useful in facilitating diagnosis of a diseaseor other condition in a patient, whether or not imaging methodology isemployed. As defined herein, a “diagnostic agent” is a type of bioactiveagent.

Diagnostic method: Any method involving an outcome aiding the medicalpractitioner in reaching a diagnosis of a clinical condition.

Dipole-dipole interaction: The attraction which can occur among two ormore polar molecules. Thus, “dipole-dipole interaction” refers to theattraction of the uncharged, partial positive end of a first polarmolecule to the uncharged, partial negative end of a second polarmolecule. Dipole-dipole interactions are exemplified by the attractionbetween the electropositive head group, for example, the choline headgroup, of phosphatidylcholine, and an electronegative atom, for example,a heteroatom, such as oxygen, nitrogen or sulphur “Dipole-dipoleinteraction” also refers to intermolecular hydrogen bonding in which ahydrogen atom serves as a bridge between electronegative atoms onseparate molecules and in which a hydrogen atom is held to a firstmolecule by a covalent bond and to a second molecule by electrostaticforces.

Electrostatic interaction: Any interaction occurring between chargedcomponents, molecules or ions, due to attractive forces when componentsof opposite electric charge are attracted to each other. Examplesinclude, but are not limited to: ionic interactions, covalentinteractions, interactions between a ion and a dipole (ion and polarmolecule), interaction between two dipoles (partial charges of polarmolecules), hydrogen bonds, i.e. hydrogen bonded to e.g. i) a nitrogenatom, an oxygen atom, or a fluor atom in one molecule, while at the sametime being bonded to ii) a nitrogen atom, an oxygen atom, or a fluoratom in another molecule or the same molecule, interchelatinginteractions, and London dispersion bonds (induced dipoles ofpolarizable molecules). Thus, for example, “ionic interaction” or“electrostatic interaction” refers to the attraction between a first,positively charged molecule and a second, negatively charged molecule.Ionic or electrostatic interactions include, for example, the attractionbetween a negatively charged bioactive agent, for example, a geneticdeterminant, and one or more of i) a positively charged lipid, forexample, a cationic lipid, ii) a positively charged saponin, for examplea cationic saponin, and iii) a positively charged sterol, for example acationic sterol.

Genetic determinant: Genetic determinant refers to nucleotides andpolynucleotides, including deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). The genetic determinant may be made by synthetic chemicalmethodology known to one of ordinary skill in the art, or by the use ofrecombinant technology, or by a combination thereof. The DNA and RNA mayoptionally comprise unnatural nucleotides or nucleotide derivativesincluding LNA (locked nucleic acids) and PNA (peptide nucleic acids),and it may be single or double stranded. “Genetic determinant” alsorefers to sense and anti-sense DNA and RNA, which are nucleotidesequences which are complementary to specific sequences of nucleotidesin DNA and/or RNA.

Hydrogen bond: An attractive force, or bridge, which may occur between ahydrogen atom which is bonded covalently to an electronegative atom, forexample, oxygen, sulfur, or nitrogen, and another electronegative atom.The hydrogen bond may occur between a hydrogen atom in a first moleculeand an electronegative atom in a second molecule (intermolecularhydrogen bonding). Also, the hydrogen bond may occur between a hydrogenatom and an electronegative atom which are both contained in a singlemolecule (intramolecular hydrogen bonding).

Hydrophobic interaction: Any interaction occurring between essentiallynon-polar (hydrophobic) components located within attraction range ofone another in a polar environment (e.g. water). As used herein,attraction range is on the scale of about 100 nm. A particular type ofhydrophobic interaction is exerted by “Van der Waal's forces”, i.e. theattractive forces between non-polar molecules that are accounted for byquantum mechanics. Van der Waal's forces are generally associated withmomentary dipole moments which are induced by neighboring molecules andwhich involve changes in electron distribution.

Immunogenic determinant: Any determinant or substance with thecapability of raising an immune response, such as a specific ornon-specific antibody response, or a cytolytic response. The immunogenicdeterminant may comprise or essentially consist of an epitope that formspart of a polypeptide and elicits an immune response when the wholepolypeptide is used as an immunogen. Such epitopes are confined to asingle or a few loci on the molecule in question.

Interaction: Transient or longer lasting attraction or binding of two ormore moieties to one another, mediated by physical forces such as e.g.electrostatic interactions and hydrophobic interactions.

Intercalation: Intercalation specifically denotes the associationbetween a contacting group and a genetic determinant wherein thecontacting group form a complex with two adjacent layers ofpurine-pyrimidine base-pairs of the genetic determinant. Theintercalating group is oriented in parallel to the base-pairs andinteracts with the genetic determinant by hydrogen bond forces,electrostatic interactions and/or hydrophobic interactions.

Iscom structure: Rigid, cage-like matrix characterised by an icosahedralsymmetry, about 30 to 40 nanometers in diameter, and composed of 12nanometer ring-like subunits.

Essentially non-polar: Nature of compounds or domains capable ofcontacting or interacting with lipids or lipophilic moieties of asimilar nature.

Lipophilic moiety: Moiety attached to or in contact with either i) anysuitable functional group of one or more compounds that is essentiallynon-polar, or ii) moiety forming an essentially non-polar domain withinthe complexes according to the present invention. Preferred lipophilicmoieties are a naturally-occurring, synthetic or semi-synthetic(modified natural) compound which is generally amphipathic. Lipidstypically comprise a hydrophilic component and a hydrophobic component.The phrase semi-synthetic (modified natural) denotes a natural compoundthat has been chemically modified in some fashion. Lipids are alsoreferred to herein as “stabilizing materials” or “stabilizingcompounds.”

Liposome: A generally spherical or spheroidal cluster or aggregate ofamphipathic compounds, including lipophilic moieties, typically in theform of one or more concentric layers, for example, monolayers, bilayersor multi-layers. They may also be referred to herein as lipid vesicles.The liposomes may be formulated, for example, from ionic lipids and/ornon-ionic lipids. Liposomes formulated from non-ionic lipids may bereferred to as niosomes. Liposomes formulated, at least in part, fromcationic lipids or anionic lipids may be referred to as cochleates.

Patient: Any member, individual, or “animal body” of the subphylumchordata, including, without limitation, mammals such as cattle, sheep,pigs, goats, horses, and humans, preferably humans; domestic animalssuch as dogs and cats; and birds, including domestic, wild and gamebirds such as cocks and hens including chickens, turkeys and othergallinaceous birds. The term also covers fish, and it does not denoteany particular age. Thus, both adult and newborn animals are intended tobe covered.

Phospholipid; Any moiety consisting of a glycerol backbone, ahydrophobic part comprising a phosphate group, and a lipophilic part inthe form of two fatty acids.

Polynucleotide: A molecule comprising at least two nucleic acids. Thenucleic acids may be naturally occurring, or locked nucleic acids (LNA),or peptide nucleic acids (PNA). Polynucleotide as used herein generallypertain to

i) a polynucleotide comprising a predetermined coding sequence, or

ii) a polynucleotide encoding a predetermined amino acid sequence, or

iii) a polynucleotide encoding a fragment of a polypeptide encoded bypolynucleotides (i) or (ii), wherein said fragment has at least onepredetermined activity as specified herein; and

iv) a polynucleotide the complementary strand of which hybridizes understringent conditions with a polynucleotide as defined in any one of (i),(ii) and (iii), and encodes a polypeptide, or a fragment thereof, havingat least one predetermined activity as specified herein; and

v) a polynucleotide comprising a nucleotide sequence which is degenerateto the nucleotide sequence of polynucleotides (iii) or (iv);

or the complementary strand of such a polynucleotide.

Polypeptide: A molecule comprising at least two amino acids. The aminoacids may be natural or synthetic.

Positively charged moiety: Any moiety comprising a positive electricalcharge capable of attracting a negative electrical charged moiety.Positively charged moieties are typically found in cationic species, oneof which is a species comprising at least one positively charged moietyat pH=7.0.

Quil A: Quillajabark Araloside A, a crude saponin mixture containingQA-7, QA-17, QA-18, and QA-21 as well as other saponins.

Region of a patient: A particular area or portion of the patient and insome instances to regions throughout the entire patient. Exemplary ofsuch regions are the pulmonary region, the gastrointestinal region, thecardiovascular region (including, myocardial tissue), the renal regionas well as other bodily regions, tissues, lymphocytes, receptors, organsand the like, including the vasculature and circulatory system, and aswell as diseased tissue, including cancerous tissue. “Region of apatient” includes, for example, regions to be treated with a bioactiveagent, regions to be targeted for the delivery of a bioactive agent, andregions to be imaged with diagnostic imaging. The “region of a patient”is preferably internal, although, if desired, it may be external. Thephrase “vasculature” denotes blood vessels (including arteries, veinsand the like). The phrase “gastrointestinal region” includes the regiondefined by the esophagus, stomach, small and large intestines, andrectum. The phrase “renal region” denotes the region defined by thekidney and the vasculature that leads directly to and from the kidney,and includes the abdominal aorta.

Receptor: A molecular structure within a cell or on the surface of acell which is generally characterized by the selective binding of aspecific substance. Exemplary receptors include cell-surface receptorsfor peptide hormones, neurotransmitters, antigens, complement fragments,immunoglobulins and cytoplasmic receptors.

Saccharide: Sugar part of saponins according to the present invention

Saponin: Any compound comprising a saccharide part linked by means of aglycosidic bond to one of i) a triterpene aglycone part, ii) a steroidaglycone part, and iii) a steroid alkaloid aglycone part. Saponin asused herein denotes either a substantially purified saponin, or one ormore saponins comprised in a crude composition or a composition obtainedby predetermined purification means. Saponin shall also denote anybiologically active fragment of any saponin. The saponins pertaining tothe present invention may be naturally occurring or synthetic, or theymay be made by chemical synthesis, or enzymatic synthesis involving oneor more enzyme catalysed steps, either in vitro or in vivo. Firstsaponins are generally incapable of forming an association with agenetic determinant as defined herein, whereas such an association isformed between second saponins and the genetic determinant.

Second saponin: Second saponins may be either anionic, neutral, orcationic. Second saponins are capable of forming an electrostaticinteraction and/or a hydrophobic interaction with a bioactive agent,including a genetic determinant. The term cationic saponin shall denotea saponin carrying a net positive charge at pH 7.0. Second saponins arepreferably cationic saponins. Alternatively, second saponins preferablycomprises at least one moiety carrying a positive charge at pH 7.0regardless that the overall net charge is not positive. Accordingly,second saponins preferably comprise at least one positively chargedmoiety or reactive group at pH=7.0, and this positively charged moietyor reactive group is according to one preferred embodiment of theinvention capable of contacting a bioactive group, including a geneticdeterminant, by means of an electrostatic interaction. In anotherembodiment, a second saponin preferably comprises an uncharged moiety ornon-polar reactive group capable of contacting a bioactive group,including a genetic determinant, by means of a hydrophobic interaction.One group of preferred second saponins are saponins comprising as theaglycone part a synthetic or naturally occurring, cationic quillaicacid, or a quillaic acid comprising at least one positively chargedgroup at pH=7.0.

Sterol: Any sterol, including any derivative thereof, comprising thecharacteristic skeleton structure of a steroid as depicted herein belowin the form of gonane. All steroids are related to a characteristicmolecular structure composed of 17 carbon atoms arranged in four ringsconventionally denoted by the letters A, B, C, and D and bonded to 28hydrogen atoms.

This skeleton structure (1) is named gonane and often referred to as thesteroid nucleus. This skeleton structure may be modified in apractically unlimited number of ways by removal, replacement, oraddition of a few atoms, moieties or reactive groups at a time. Theskilled artisan will know how to isolate steroids from plants andanimals, and optionally prepare derivatives by chemical and/or enzymatictreatment of natural steroids. The skilled artisan will also know how tosynthesize synthetic steroids from simpler compounds, or naturalprecursors or parts thereof, and optionally prepare derivatives of suchcompounds by chemical and/or enzymatic treatment of steroids thusobtained. Accordingly, one preferred steroid compound according to theinvention is a sterol and any cationic derivative thereof, in particularcationic derivatives obtained by linking a cationic moiety, or acationic reactive group, to e.g. an OH-group of the sterol including anOH-group located at position 3 of the steroid skeleton, including theOH-group of cholesterol located at position 3 (C:3, or 3-OH).Consequently, preferred sterols according to the present invention arecholesterol (CAS (Chemical Abstract) accession no. 57-88-5) and anycationic derivative thereof, in particular cationic derivatives obtainedby linking a cationic moiety, or a cationic reactive group, to theOH-group located at position 3 of the steroid skeleton (C3, or 3-OH).Preferred examples of such compounds are illustrated in FIG. 5. Firststerols are generally incapable of forming an association with a geneticdeterminant as defined herein, whereas such an association is formedbetween second sterols and the genetic determinant.

Second sterol: Second sterols may be either anionic, neutral, orcationic. Second sterols are capable of forming an electrostaticinteraction and/or a hydrophobic interaction with a bioactive agent,including a genetic determinant. The term cationic sterol shall denote asterol carrying a net positive charge at pH 7.0. Second sterols arepreferably cationic sterols. Alternatively, second sterols preferablycomprises at least one moiety carrying a positive charge at pH 7.0regardless that the overall net charge is not positive. Accordingly,second sterols preferably comprise at least one positively chargedmoiety or reactive group at pH=7.0, and this positively charged moietyor reactive group is according to one preferred embodiment of theinvention capable of contacting a bioactive group, including a geneticdeterminant, by means of an electrostatic interaction. In anotherembodiment, the second sterol preferably comprises an uncharged moietyor non-polar reactive group, and this uncharged moiety or non-polarreactive group is according to another preferred embodiment of theinvention capable of contacting a bioactive group, including a geneticdeterminant, by means of a hydrophobic interaction.

Steroid alkaloid glycoside: Saponin comprising a saccharide part linkedby means of a glycosidic bond to a steroid alkaloid aglycone part.

Steroid glycoside: Saponin comprising a saccharide part linked by meansof a glycosidic bond to a steroid aglycone part.

Stringent conditions: Stringent conditions as used herein shall denotestringency as normally applied in connection with Southern blotting andhybridization as described e.g. by Southern E. M., 1975, J. Mol. Biol.98; 503-517. For such purposes it is routine practise to include stepsof prehybridization and hybridization. Such steps are normally performedusing solutions containing 6×SSPE, 5% Denhardt's, 0.5% SDS 50%formamide, 100 μg/ml denaturated salmon testis DNA (incubation for 18hrs at 42° C.), followed by washings with 2×SSC and 0.5% SDS (at roomtemperature and at 37° C.), and a washing with 0.1×SSC and 0.5% SDS(incubation at 68° C. for 30 min), as described by Sambrook et al.,1989, in “Molecular Cloning/A Laboratory Manual”, Cold Spring Harbor),which is incorporated herein by reference.

Substantially pure saponin: Saponin substantially free from compoundsnormally associated with the saponin in its natural state, wherein thesaponin exhibits a constant and reproducible chromatographic responseand/or elution profile and/or biologic activity. The term “substantiallypure” as used herein is not meant to exclude artificial or syntheticmixtures of the saponin with other compounds. Preferably, thesubstantially pure saponin is purified to one or more of the followingstandards: i) Appearing as only one major carbohydrate staining band onsilica gel TLC (EM Science HPTLC Si60) in a solvent system of 40 mmacetic acid in chloroform/methanol/water (60/45/10, v/v/v), ii)Appearing as only one major carbohydrate staining band on reverse phaseTLC (EM Science Silica Gel RP-8) in a solvent system of methanol/water(70/30, v/v), 3) Appearing as only one major peak upon reverse-phaseHPLC on Vydac C4 (5 μm particle size, 330 Ångstrøm pore, 4.6 mm ID×25 cmL) in 40 mM acetic acid in methanol/water (58/42, v/v).

Targeting ligand: Any material or substance which, when comprised in acomplex according to the invention, is capable of promote targeting oftissues and/or receptors in vivo or in vitro with the complexes of thepresent invention, or compositions such as e.g. biodegradablemicrosphere or liposomes comprising such complexes. In the latter casethe compositions comprise the targeting ligand independently of whetheror not the complexes also comprise the targeting ligand. The targetingligand may be synthetic, semi-synthetic, or naturally-occurring.Materials or substances which may serve as targeting ligands include,for example, proteins, including antibodies, antibody fragments,hormones, hormone analogues, glycoproteins and lectins, peptides,polypeptides, amino acids, sugars, saccharides, includingmonosaccharides and polysaccharides, carbohydrates, vitamins, steroids,steroid analogs, hormones, cofactors, and genetic determinants,including nucleosides, nucleotides, nucleotide acid constructs,polynucleotides, optionally constructs or polynucleotides comprisingderivatised nucleotides such as PNA (peptide nucleic acid) and/or LNA(locked nucleic acid). A “precursor” to a targeting ligand refers to anymaterial or substance capable of being converted to a targeting ligand.Such conversion may involve, for example, anchoring a precursor to atargeting ligand. Exemplary targeting precursor moieties includemaleimide groups, disulfide groups, such as ortho-pyridyl disulfide,vinylsulfone groups, azide groups, and α-iodo acetyl groups.

Therapeutic agent: The terms “pharmaceutical agent” or “drug” or“medicament” refers to any therapeutic or prophylactic agent which maybe used in the treatment (including the prevention, diagnosis,alleviation, or cure) of a malady, affliction, condition, disease orinjury in a patient. Therapeutically useful genetic determinants,peptides, polypeptides and polynucleotides may be included within themeaning of the term pharmaceutical or drug. As defined herein, a“therapeutic agent,” “pharmaceutical agent” or “drug” or “medicament” isa type of bioactive agent.

Tissue: Specialized cells which may perform a particular function. Theterm “tissue” may refer to an individual cell or a plurality oraggregate of cells, for example, membranes or organs. The term “tissue”also includes reference to an abnormal cell or a plurality of abnormalcells. Exemplary tissues include pulmonary tissue, myocardial tissue,including myocardial cells and cardiomyocytes, membranous tissues,including endothelium and epithelium, laminae, blood, connective tissue,including interstitial tissue, and tumors. “Cell” refers to any one ofthe minute protoplasmic masses which make up organized tissue,comprising a mass of protoplasm surrounded by a membrane, includingnucleated and unnucleated cells and organelles.

Treatment: Method involving therapy including surgery of a clinicalcondition in an individual including a human or animal body. The therapymay be profylactic, ameliating or curative.

Triterpene glycoside: Saponin comprising a saccharide part linked bymeans of a glycosidic bond to a triterpene aglycone part.

Zeta potential: The parameter of zeta potential is a measure of themagnitude of the repulsion or attraction between particles. Itsmeasurement relates to some extent to the overall charge of particlesbut also to the stability of particles in dispersion. The surface chargeof particles in polar liquids dose not directly correlate to theelectrical potential at the surface of the particle but to the potentialthat in the close vicinity of the particle.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to complexes which are capable ofbinding bioactive agents such as e.g. genetic determinants such as forexample polynucleotides, therapeutic agents, e.g. polypeptides,diagnostic agents and imaging agents. When attached to the complexesaccording to the invention, the bioactive agents are capable of beingtaken up by a biological cell including a human or animal cell. Thecomplexes according to the invention are thus useful as a means fortransfecting a biological cell with e.g. a polynucleotide either in vivoor in vitro.

In other preferred embodiments there are provided methods of deliveringbioactive agents to a patient and/or treating conditions in a patientcomprising administering to the patient a complex according to theinvention, or a composition comprising such a complex.

The present invention describes methods of delivering bioactive agentsto a patient and/or treating conditions in a patient comprisingadministering to the patient a complex according to the invention, or acomposition comprising such a complex.

The present invention also describes methods of diagnosing the presenceof diseased tissue in a patient comprising administering to the patienta complex according to the invention, or a composition comprising such acomplex.

The present invention also describes methods of providing an image of aninternal region of a patient comprising administering to the patient acomplex according to the invention, or a composition comprising such acomplex.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the carbon skeleton of the three main classes ofsaponins according to the invention. Depending on the type of geninpresent, the saponins according to the invention can be divided intothree major classes: i) triterpene glycosides, ii) steroid glycosides,and iii) steroid alkaloid glycosides.

FIG. 2 illustrates the carbon skeleton of various pentacyclictriterpenes divided into three main classes, depending on whether theyhave a β-amyrin, α-amyrin or lupeol skeleton. In addition thereto,several minor classes also exist. The aglycone skeleton of the followingtriterpene glycosides are illustrated in the figure: Oleane (β-Amyrin),Ursane (α-Amyrin), Lupane, Taraxastane, Taraxerane, Friedelane,Glutinane, and Hopane.

FIG. 3 illustrates the olean-12-en skeleton of triterpene glycosides. Anumber of such aglycone variants pertaining to the present invention arelisted in Table 1 herein.

FIG. 4 Illustrates other representative variants of triterpene aglyconesaccording to the present invention and listed in Table 2 herein. Theaglycones of FIG. 4 are representative of aglycones which do not have anolean-12-en skeleton. Examples are listed in Table 2 herein.

FIG. 5 illustrates preferred second sterols according to the inventionand capable of being incorporated into the complexes according to thepresent invention. The listed compounds are ChoTB and ChoSC as describedby Leventis, R. et. al. (1989) Biochim Biophys Acta 1023, 124; DC-Choland TC-Chol as described by Avanti Lipids and further characterisedherein; Lipid 67 (also known as GL-67), as described by Lee, E. R. etal. (1996) Human Gene Therapy 7, 1701; BGTC is an acronym for 3-beta[N′,N′-diguanidioethyl-aminoethane)carbamoyl]cholesterol; and BGSC is anacronym for 3-beta[4N-(1N,8N-diguanidinospermidine)-carbamoyl]cholesterol.

FIG. 6 illustrates preferred lipophilic moieties according to theinvention and capable of being incorporated into the complexes accordingto the present invention. The listed compounds are DOTIM, or1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl)-3-(2-hydroxy-ethyl)-imidazoliniumchloride,DODAC is an acronym for dioleoyidimethylammonium chloride, and GAP-DLRIEis an acronym for(+/−)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide.

FIG. 7 illustrates yet further preferred lipophilic moieties capable ofbeing incorporated into the complexes according to the presentinvention. The listed compounds are DOTMA, orN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE,or N,N-dimethyl-1,2-dimyristoyloxy-3-aminopropane, DOSPA, or2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammoniumtrifluoroacetate, and DOGS, or Transfectam®, as described by Behr, J. P.et al. (1989) Proc, Natl. Acad. Sci. USA 86,6982, U.S. Pat. No.5,171,678.

FIG. 8 Illustrates even further preferred lipophilic moieties capable ofbeing incorporated into the complexes according to the presentinvention. The listed compounds are DOTAP, or(N[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium-ethylsulfate;Tfx-50, orN,N,N′,N′-tetramethyl-N,N-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butaned-iammoniumiodide;DDAB, or dimethyl-dioctadecylammonuim-bromide; and TM-TPS, ortetrapalmitylspermine,

FIG. 9 illustrates the influence of organic solvent on the formation ofISCOM-matrix as evaluated by visual inspection by electron microscopy(EM). The primary criterion was the shape and uniformity of thestructures, secondary the number of ISCOM particles. The formation ofISCOM-matrix was unaffected by the presence of DMSO up to aconcentration of 20% v/v as shown in panel A. Above this limit the yieldwas affected. With 25% DMSO intact structures could be observed but thenumber of ISCOMs were reduced. The same pattern was observed for DMF, asdescribed in detail in Example 1 herein. Panel B in FIG. 9 demonstratesthat EtOH was compatible with the process at concentrations up toapprox. 15%. Some diverging structures appeared among intact structuresprepared at this concentration.

FIG. 10 illustrates that undisrupted ISCOM complexes according to thepresent invention protect embedded peptides from hydrolysis during theharsh conditions of the acidic hydrolysis during an amino acid analysis.This result emphasizes the high stability of the ISCOM structure and thedifference in chemical environment within ISCOMs.

FIG. 11 illustrates measurement of zeta potentials for ISCOMs containingno DC-cholesterol performed as described in example 4. Similar to theexperiments described in FIG. 12, the Zeta-potential was measured fivetimes. The average Zeta-potential was found to be approx. −50 mV (seetable below), which is significantly different from the ISCOMscontaining DC-cholesterol (DC-cholesterol to cholesterol ratio 1:1 or1:2), as demonstrated in FIG. 12.

Recorded mean Conductivity value (mV) Width (mV) (mS/cm) Title 16 −51.66.3 0.300 Std. ISCOM 17 −45.0 6.2 0.362 Std. ISCOM 18 −51.7 6.1 0.215Std. ISCOM 19 −50.8 6.3 0.379 Std. ISCOM 20 −45.9 6.3 0.317 Std. ISCOM

FIG. 12 illustrates measurements of zeta potentials for modified ISCOMscomprising a second sterol in the form of DC-cholesterol. TheDC-cholesterol to cholesterol ration was 1:1 (50% substitution). Eachcurve represents one of five measurements. The Zeta-potential was closeto 0 mV with some variation between measurements (see table below). WhenISCOMs with a DC-cholesterol to cholesterol of 1:2 (25%) were measuredan average Zeta-potential of approx. −25 mV observed (data not shown).

Recorded mean Conductivity value (mV) Width (mV) (mS/cm) Title 11 2.06.4 0.172 DC-CH 50% 12 6.0 6.5 0.182 DC-CH 50% 13 0.5 6.2 0.216 DC-CH50% 14 6.7 6.6 0.169 DC-CH 50% 15 5.0 6.4 0.159 DC-CH 50%

Saponins

Saponins pertaining to the present invention are described in detailherein below. Saponins are glycosidic compounds which comprises anaglycone compound and a saccharide compound linked together by aglycosidic bond. The asymmetric distribution of their hydrophobic(aglycone) and hydrophilic (saccharide) moieties confers an amphipathiccharacter to the saponins according to the present invention.

Saponins are produced by many organisms as secondary metabolites. Theyare widely distributed among higher plants and in some marineinvertebrates. Plant material often contains triterpene saponins inconsiderable amounts. Thus, primula root contains about 5-10% saponin,licorice root between 2% and 12% glycyrrhizin, quillaia bark up to 10%of a saponin mixture and the seeds of the horse chestnut up to 13%aescine. In other words, the concentration of saponins in plants is highwhen compared with other secondary metabolites.

The aglycone or non-saccharide portion of the saponin molecule is calledthe genin or sapogenin. Depending on the type of genin present, thesaponins can be divided into three major classes, i) triterpeneglycosides, ii) steroid glycosides, and iii) steroid alkaloid glycosides(FIG. 1).

In addition to saponins comprising triterpene saponins, the presentinvention also pertains to saponins comprising steroid sapogeninsderived from a furostan skeleton or a spirostan skeleton. The presentinvention further pertains to saponins comprising steroid alkaloidsapogenins derived from a solanidan skeleton or a spirosolan skeleton.The steroid alkaloid glycosides, or glycoalkaloids, share many physicaland biological properties with steroid glycosides, but alkaloidglycosides are usually considered separately because their steroidalstructure contains nitrogen.

Saponins Comprising a Triterpene Glycoside

Triterpene glycosides represent one preferred class of saponinsaccording to the present invention. The pentacyclic triterpenes can bedivided into three main classes, depending on whether they have aβ-amyrin, α-amyrin or lupeol skeleton. In addition thereto, severalminor classes also exist as illustrated in FIG. 2.

According to the present invention, saponins in the form of triterpeneglycosides preferably comprises an aglycone skeleton selected from thegroup of compounds consisting of Oleane (β-Amyrin), Ursane (α-Amyrin),Lupane, Taraxastane, Friedelane, Glutinane, Hopane, Dammarane,Lanostane, Holostane, and Cycloartane.

The triterpene aglycone may be hydroxylated at C-3 and certain methylgroups may be oxidized to hydroxymethyl, aldehyde or carboxylfunctionalities. When an acid moiety is esterified to the triterpeneaglycone, the term ester saponin is used for the respective glycosides.Further important structural elements of this class is: The unsaturationat C-12(13); the functionalization of the methyl group of C-28, C-23 orC-30; and polyhydroxylation at C-2, C-7, C-11, C-15, C-16, C-19. Theformation of an additional ring structure is possible throughetherification or lactonization, and esterification by aliphatic acidsis also possible.

There are numerous structural variants of the triterpene glycoside classof saponins comprising a oleanane skeleton (olean-12-en skeleton). Anumber of such aglycone variants pertaining to the present invention arelisted in Table 1 herein below. Their skeleton is illustrated in FIG. 3.

Other representative variants of triterpene aglycones according to thepresent invention are listed in Table 2. The aglycones of Table 2 arerepresentative of aglycones which do not have an olean-12-en skeleton.Examples of such skeletons are illustrated in FIG. 4

A review article by Tschesche and Wulff (1972), incorporated herein byreference, gives further references and examples, together with physicalconstants, of both oleananes and other triterpenes. Mahato andco-workers (Das and Mahato, 1983; Mahato et al. 1992; both of which areincorporated herein by reference) have published lists of recentlyisolated triterpenes (not necessarily from saponins) with their physicalconstants and plant sources.

Oleanane triterpenes (and some of their glycosides) have been thesubject of an update (Mallavarapu, 1990: incorporated herein byreference), covering various aspects of their occurrence and chemistry.Another authoritative source of information on the triterpenes is thebook written by Boiteau and colleagues (1964; incorporated herein byreference).

TABLE 1 Structures of commonly occurring olean-12-en aglycones No.Olean-12-en aglycone —OH ═O —COOH Other 1 β-Amyrin 3β 2 Oleanolic acid3β 28 3 Epikatonic acid 3β 29 4 α-Boswellic acid 3α 24 5 Momordic acid3β 1 28 6 Glycyrrhetinic acid 3β 11 30 7 Gypsogenin 3β 23 28 8Gypsogenic acid 3β 23,28 9 Cincholic acid 3β 27,28 10 Serjanic acid 3β28 30-COOMe (30-O-methyl-spergulagenate) 11 Maniladiol 3β,16β 12Sophoradiol 3β,22β 13 3β,22β-Dihydroxyolean-12-en-29-oic acid 3β,22β 2914 2β-Hydroxyoleanolic acid 2β,3β 28 15 Maslinic acid 2α,3β 28 16Echinocystic acid 3β,16α 28 17 Hederagenin 3β,23 28 18 Phytolaccagenicacid 3β,23 28 19 Siaresinolic acid 3β,19α 28 20 21β-Hydroxyoleanolicacid 3β,21β 28 (machaerinic acid) 21 29-Hydroxyoleanolic acid 3β,29 2822 Azukisapogenol 3β,24 29 23 Soyasapogenol E 3β,24 22 24 Primulagenin D3β,16α 28 (28-dehydroprimulagenin) 25 3β,24-Dihydroxyolean-12,15- 3β,2415-en dien-28-oic acid 26 Soyasapogenol C 3β,24 21-en 27 Glabrinic acid3β,26 11 30 28 Quillaic acid 3β,16 23 28 29 21β-Hydroxy-gypsogenin 3β,2123 28 30 Barringtogenic acid 2α,3β 23,28 31 Medicagenic acid 2β,3β 23,2832 Dianic acid 3β,29 23,28 33 Soyasapogenol B 3β,22β,24 343β,22β,24-Trihydroxy- 3β,22β,24 29 olean-12-en-29-oic acid 35Primulagenin A 3β,16α,28 36 2β,3β,28-Trihydroxy-olean-12-en 2β,3β,28 37Priverogenin A 3β,16α,22α 28 38 16a-Hydroxy- 3β,16α,23 28 hederagenin(caulophyllogenin) 39 21β-Hydroxy-hederagenin 3β,21β,23 28 403β,21β,22β-Trihydroxy- 3β,21β,22β 29 olean-12-en-29-oic acid 4123-Hydroxyimberbic acid 1α,3β,23 29 42 Arjunic acid 2α,3β,19α 28 43Arjunolic acid 2α,3β23 28 44 Asterogenic acid 2β,3β,16α 28 45 Bayogenin2β,3β,23 28 46 16-Hydroxy-medicagenic acid 2β,3β,16 23,28 47Presenegenin 2β,3β,27 23,28 48 Jaligonic acid 2β,3β,23 28,30 49Phytolaccagenin 2β,3β,23 28 30-COOMe 50 Belleric acid 2α,3β,23,24 51Barringtogenol A 2α,3β,23,28 52 Protobassic acid 2β,3β,6β,23 28 53Platycogenic acid C 2β,3β,16β,21β 28 54 Polygalacic acid 2β,3β,16α,23 2855 Tomentosic acid 2α,3β,19β,23 28 56 Arjungenin 2α,3β,19β,23 28 57Esculentagenic acid 2β,3β,23,30 58 23-Hydroxylongispinogenin3β,16β,23,28 59 Cyclamiretin E 3β,16α,28,30 60 Soyasapogenol A3β,21β,22β,24 61 Oxytrogenol 3β,22β,24,29 62 3α,21β,22α,28-3α,21β,22α,28 Tetrahydroxyolean-12-en 63 3β,23,27,29- 3β,23,27,29 28Tetrahydroxyoleanolic acid 64 Barringtogenol C 3β,16α,21β,22α,28 65Camelliagenin C 3β,16α,22α,23,28 66 16α-Hydroxyprotobassic acid2β,3β,6β,16α,23 28 67 Platycodigenin 2β,3β,16α,23,24 28 68Protoaescigenin 3β,16α,21β,22α,24,28 69 Theasapogenol A3β,16α,21β,22α,23,28 70 R₁-Barrigenol 3β,15α,16α,21β,22α,28

TABLE 2 Triterpene aglycones (other than olean-12-en type). No. NameSkeleton —OH ═O —COOH Other 71 Protoprimulagenin A A 3β,16α 72Cyclamiretin A A 3β,16α 30 73 Rotundiogenin A A 3β,16α 11-en 74Saikogenin E A 3β,16α 11-en 75 Anagalligenone A 3β,23 16 76 Saikogenin FA 3β,16β,23 77 Saikogenin G A 3β,16α,23 (anagalligenin B) 78Priverogenin B A 3β,16α,22α 79 Anagalligenin A A 3β,16α,22α,28 80α-Amyrin B 3β 12-en 81 Ursolic acid B 3β 28 12-en 82 Quinovic acid B 3β27,28 12-en 83 3β-Hydroxyurs-12,20(30)-dien- B 3β 27,28 12,20(30)-dien27,28-dioc acid 84 Pomolic acid B 3β,19α 28 12-en 85 Ilexgenin B B3β,19α 28 12-en 86 Ilexgenin A B 3β,19α 24,28 12-en 8721β-Hydroxyursolic acid B 3β,21β 28 12-en 88 23-Hydroxyursolic acid B3β,23 28 12-en 89 3β,23-Dihydroxy- B 3β,23 28 20-en taraxer-20-en-28-oicacid 90 Rotundic acid B 3β,19α,23 28 12-en 91 Rotungenic acid B3β,19α,24 28 12-en 92 Madasiatic acid B 2α,3β,6β 28 12-en 93 Asiaticacid B 2α,3β,23 28 12-en 94 Euscaphic acid B 2α,3α,19α 28 12-en 95Tormentic acid B 2α,3β,19α 28 12-en 96 2β,3β,19α-Trihydroxyurs-12- B2α,3α,19α 23,28 12-en en-23,28-dioic acid 97 6β-Hydroxytormentic acid B2α,3β,6β,19α 28 12-en 98 7α-Hydroxytormentic acid B 2α,3β,7α,19α 2812-en 99 23-Hydroxytormentic acid B 2α,3β,19α,23 28 12-en 10024-Hydroxytormentic acid B 2α,3β,19α,24 28 12-en 1011α,3β,19α,23-Tetrahydroxyurs-12- B 1α,3β,19α,23 28 12-en en-28-oic acid102 Madecassic acid B 2α,3β,6β,23 28 12-en 103 6β,23-Dihydroxytormenticacid B 2α,3β,6β,19α,23 28 12-en 104 Lupeol C 3β 20(29)-en 105 Betulin C3β,28 20(29)-en 106 Betulinic acid C 3β 28 20(29)-en 107 3-epi-Betulinicacid C 3α 28 20(29)-en 108 3β,23-Dihydroxylup- C 3β,23 28 20(29)-en20(29)-en-oic acid 109 3α-Hydroxylup- C 3α 23,28 20(29)-en20(29)-en-23,28-dioic acid 110 3α,11α-Dihydroxylup- C 3α,11α 23,2820(29)-en 20(29)-en-23,28-dioic acid 111 Cylicodiscic acid C 3β,27α 2820(29)-en 112 Mollugogenol B D 3β,6α 15,17(21)-dien 113(20S)-Protopanaxadiol E 3β,12β,20S 24-en 114 (20S)-Protopanaxatriol E3β,6α,12β,20S 24-en 115 Bacogenin A₁ E 3β,19,20 16 24-en 116Seychellogenin F 3β 7,9(11)-dien 18,20-lactone 117 Mollic acid G 1α,3β28 118 3β,21,26-Trihydroxy-9,19- G 3β,21,26 24-en cyclolanost-24-en 119Thalicogenin G 3β,16β,22,28 24-en 120 3β,16β,24,25-Tetrahydroxy-9,19- G3β,16β,24,25 cyclolanostane 121 3β,6α,16β,24,25-Pentahydroxy- G3β,6α,16β,24,25 9,19-cyclolanostane 122 Cycloastragenol(astramembrangenin, H 3β,6α,16β,25 cyclosiversigenin) 1233β-Hydroxy-9,19- I 3β 24(28)-en cyclolanost-24(28)-en 124 Jessic acid I1α,3β 23 29 24(28)-en The skeletons are designated by capital letters Ato I as indicated in FIG. 4.

The complexes according to the present invention have molecular weightsranging from for example about 400 daltons to more than 2,000 daltons.Further examples are from about 500 daltons, such as from about 600daltons, for example from about 700 daltons, such as from about 800daltons, such as from about 900 daltons, for example from about 1000daltons, such as from about 1100 daltons, such as from about 1200daltons, for example from about 1300 daltons, such as from about 1400daltons, such as from about 1500 daltons, for example from about 1600daltons, such as from about 1700 daltons, such as from about 1800daltons, for example from about 1900 daltons, such as from about 2000daltons, to preferably less than 4,000 daltons.

In one embodiment of the present invention, the saponin compound isacylated with one or more organic acids such as acetic acid, malonicacid, angelic acid and the like (see fx Massiot, G. & Lavaud, C., Stud.Nat. Prod. Chem. 15:187-224 (1995), incorporated herein by reference).

Saccharide Moieties of Saponins

Saponins according to the present invention, including the aglycone asillustrated in Table 1 and Table 2 herein above, have one or more linearor branched saccharide chains attached to the aglycone part via aglycosidic ether or ester bond.

According to the number of saccharide chains attached to the aglycone,the saponins can be monodesmosidic saponins (with a single saccharidechain), or bidesmosidic saponins (with two saccharide chains).

In the monodesmosidic saponins according to the invention, thesaccharide chain is preferably attached by a glycosidic ether linkage atthe C-3 of the aglycone. In addition to the C3 linked saccharide chain,bidesmosidic saponins have a second saccharide chain bound at C-28(triterpene saponins) or at C-26 (steroid saponins) by an ester linkage.Because of the typical lability of esters, bidesmosidic saponins arereadily converted into their monodesmosidic forms by mild hydrolysis(Hostettmann, K., et al., Methods Plant Biochem. 7:435-471 (1991)).

Bidesmosidic saponins according to the invention preferably have twosugar chains, one of which may be attached through an ether linkage atC-3, and one attached through an ester linkage (acyl glycoside) at C-28(triterpene saponins), or an ether linkage at C-26 (furostanolsaponins).

Bidesmosidic saponins are easily converted into monodesmosidic saponinsby, for example, hydrolysis of the esterified sugar at C-28 intriterpene saponins, and they differ from monodesmosidic saponins withrespect to some properties and activities. Also, when one sugar chain isattached at C-3, a second sugar group may be esterified to the carboxylgroup at C-17 of the aglycone. Furthermore, some dammarane glycosidesand lanostane glycosides may have a second or even a thirdglycosidically bound sugar chain.

Tridesmosidic saponins according to the invention have three sugarchains. Hydrolysis of the esterified sugars result in conversion intobidesmosidic saponins and/or monodesmosidic saponins. An example of onetridesmosidic triterpene is a 9,19-cyclolanostane (cycloartane)substituted glycosidically at positions C-3, C-6 and C-25. An example ofa tridesmosidic olean-12-en saponin is quinoside A, in which sugars areattached at positions C-3, C-23 and C-28 of hederagenin (Meyer et al.1990). Also, a tridesmoside of 16α-hydroxymedicagenic acid (zahnic acid)has been found in the aerial parts of alfalfa (Medicago saliva,Leguminosae) (Oleszek et al. 1992).

The saccharide moiety of saponins according to the invention may belinear or branched, with about 11 being the highest number ofmonosaccharide units yet found in a saponin (Clematoside C from Clematismanshurica (Ranunculaceae); Khorlin et al. 1965). However, the presentinvention is not limited to saccharide moieties containing 11 or lessmonosaccharide units. Saponins according to the present invention maycomprise less than 10 sacchardde moieties, such as less than 9saccharide moieties, for example less than 8 saccharide moieties, suchas less than 7 saccharide moieties, for example less than 6 saccharidemoieties, such as less than 5 saccharide moieties, for example less than4 saccharide moieties, such as less than 3 saccharide moieties, forexample less than 2 saccharide moieties, such as one saccharide moiety.

In fact, most saponins so far isolated tend to have relatively short(and often unbranched) sugar chains, containing from about 2 to about 5monosaccharide residues. Kochetkov and Khorlin (1966) have introducedthe term oligoside for glycosides containing more than 3 to 4monosaccharides.

Accordingly, there are also provided saponins according to the presentinvention comprising one or more sugar chains, for example two or threesugar chains, wherein one or more sugar chains comprises for examplefrom about 2 to about 11 monosaccharide residues, such as from about 2to about 10 monosaccharides, for example from about 2 to about 11monosaccharide residues, such as from about 2 to about 10monosaccharides, for example from about 2 to about 9 monosaccharideresidues, such as from about 2 to about 8 monosaccharides, for examplefrom about 2 to about 7 monosaccharide residues, such as from about 2 toabout 6 monosaccharides, for example from about 2 to about 5monosaccharide residues, such as from about 2 to about 4monosaccharides, for example from about 2 to about 3 monosaccharideresidues.

In another embodiment the present invention provides saponins comprisingone or more sugar chains, for example two or three sugar chains, whereinone or more sugar chains comprises more than 3 monosaccharides, such asmore than 4 monosaccharides, for example more than 5 monosaccharides,such as more than 6 monosaccharides, for example more than 7monosaccharides, such as more than 8 monosaccharides, for example morethan 9 monosaccharides, and preferably less than 11 monosaccharideresidues.

Oligosides as used herein refer to saponin glycosides containing 4 ormore monosaccharides, such as more than 5 monosaccharides, for examplemore than 6 monosaccharides, and independently thereof preferably lessthan 12 monosaccharides, such as less than 11 monosaccharides, forexample less than 10 monosaccharides, such as less than 9monosaccharides, for example less than 8 monosaccharides.

However, saponins according to the present invention may also compriseone or more sugar chains, for example two or three sugar chains, whereinone or more of said sugar chains comprises more than 11 monosaccharides,for example about 15 monosaccharides, such as about 20 monosaccharides,for example more than about 25 monosaccharides, such as more than about40 monosaccharides.

Preferred monosaccharide residues of saponin glycosides according to thepresent invention are: D-glucose (Glc), D-galactose (Gal), D-glucuronicacid (GlcA), D-galacturonic acid (GalA), L-rhamnose (Rha), L-arabinose(Ara), D-xylose (Xyl) and D-fucose (Fuc). Also preferred are D-apiose(Api), D-ribose (Rib), and D-allose (All). Furthermore, saponinsobtained from marine organisms often contain D-quinovose (Qui)(sometimes written as D-chinovose). All the above abbreviations are usedin accordance with IUPAC recommendations (Pure Appl. Chem. (1982). vol.54, p. 1517-1522)

In addition to the above-mentioned monosaccharides the present inventionalso pertains to unusual monosaccharides such as uronic acids that areknown to occur in some triterpene glycosides. Another example of unusualmonosaccharides are monosaccharides comprising an amino saccharideand/or an acylated saccharide.

Among the preferred monosaccharides directly attached to the saponinaglycone are glucose, arabinose, glucuronic acid and xylose. Suchmonosaccharides thus forms the link between the saccharide part and theaglycone part of the saponin.

Another group of saccharides according to the invention are saccharidescomprising acylated sugar moieties, as well as saccharides comprisingmethylated and/or sulphated sugar moieties.

In accordance with generally agreed nomenclature, the configurations ofthe interglycosidic linkages are given herein by a and A, respectively,and the monosaccharides making up the sugar part of saponins accordingto the invention may adopt a pyranose (p) and/or a furanose (f) form.

Quillaja Saponins

As used herein, saponins from the bark of the Quillaja saponaria Molinatree are termed Quillaja saponins. Quillaja saponins represent one groupof particularly preferred saponins according to the present invention.Quillaja saponins are either first saponins or second saponins, whereinthe latter group of Quillaja saponins are capable of forming andinteraction with a genetic determinant.

Quillaja saponins are found as a mixture of about twenty structurallyclosely related triterpenoid glycosides with minimal differences betweenthem (Higuchi, R. et al., Phytochemistry 26:229 (1987); ibid., 26:2357(1987); ibid., 27:1169 (1988); Kensil et al., U.S. Pat. No. 5,057,540(1991); Kensil et al., Vaccines 92:35 (1992)). Quillaja saponins arechemically and immunologically well-characterized (see fx Dalsgaard, K.Arch. Gesamte Virusforsch. 44:243 (1974), Dalsgaard, K., Acta Vet.Scand. 19 (Suppl. 69):1 (1978); Higuchi, R. et al., Phytochemistry26:229 (1987); ibid. 26:2357 (1987); ibid. 27:1168 (1988); Kensil, C. etal., J. Immunol. 146:431 (1991); Kensil et al., U.S. Pat. No. 5,057,540(1991); Kensil et al., Vaccines 92:35 (1992); Bomford, R. et al.,Vaccine 10:572 (1992); Kensil, C. et al., U.S. Pat. No. 5,273,965(1993);); Kensil, C. et al., U.S. Pat. No. 5,443,829 (1995);); Kensil,C. et al., U.S. Pat. No. 5,583,112 (1996); and Kensil, C. et al., U.S.Pat. No. 5,650,398 (1997); all of which are incorporated herein byreference).

Quillaja saponins belong to a family of closely related O-acylatedtriterpene glycoside structures. They have an aglycone triterpene(quillaic acid), with branched saccharide chains attached to positions 3and 23, and an aldehyde group in position 23. A unique characteristic ofQuillaja saponins pertaining to the invention is the presence of certainacyloil acyl moieties linked at the C-3 hydroxy group of a fucopyranosebound by an ester bond to position 28 of quillaic acid. Preferred acylmoieties are 3,5-dihydroxy-6-methyloctanoic acid,3,5-dihydroxy-6-methyloctanoic acid,5-O-α-L-rhamno-pyranosyl-(1→>2)-α-L-arabino--furanoside, and5-O-α-L-arabino-furanoside.

Quillaja saponins according to the present invention may be obtainedfrom quillaja plant species including Quillaja saponaria Molina andothers either as a crude extract, or as an extract which have beenpurified by various open column techniques (i.e. chromatography by meansof e.g. ion exchange-, size exclusion-, hydrophobic-, affinity-, andotherwise). Such purified or semi-purified saponins are generallyreferred to in the art as “Quil A”, or “Quadri A”, as described by WO95109179, which is incorporated herein by reference,

The saponins may also be purified by high resolution hydrophobicinteraction techniques, such as e.g. HPLC, and this form of purificationgenerates fractions known in the art as e.g. “Quadri 1”, “Quadri2”, andthe like (see e.g. WO 95/09179, as well as Kamstrup et al. Vaccine(2000), vol 18, no. 21, 2244-2249, incorporated herein by reference).

Particularly preferred are saponin extracts from Quillaja saponariaMolina, primarily the DQ-extract produced according to K. Dalsgaard:Saponin Adjuvants, Bull. Off Int Epiz. 77 (7-8), 1289-1295 (1972), andQuil A which is produced according to K. Dalsgaard: Saponin AdjuvantsIII, Archiv fur die Gesamte Virusforschung 44, 243-254 (1974). Alsomixtures of such glycosides pertain to the present invention.

The amount of glycoside added should be at least 1-3 times theircritical micelle formation concentration (CMC), preferably at least 5,especially at least 7-12 times. Preferably Quil A is used, which has acritical micelle formation concentration of 0.03% by weight. The amountof Quil A should then be at least 0.02% by weight, especially 0.05-0.5%by weight, preferably 0.2% by weight.

Further fractions of saponins according to the present invention aredescribed in detail herein below. According to U.S. Pat. No. 5,057,540,the contents of which are incorporated herein by reference, saponins canbe purified from an aqueous extract of the bark of the South Americantree, Quillaja saponaria Molina. At least 22 peaks with saponin activitywere separable.

The predominant purified Quillaja saponins are QA-7, QA-17, QA-18, andQA-21. These saponins have been purified by high pressure liquidchromatography (HPLC) and low pressure silica chromatography. QA-21 canbe further purified using hydrophilic interaction chromatography (HILIC)and resolved into two peaks, QA-21-V1 and QA-21-V2, that are differentcompounds.

Thus, “QA-21” designates the mixture of components QA-21-V1 and QA-21-V2that appear as a single peak on reversed-phase HPLC on VYDAC C4 (5 μmparticle size, 330 Ångstrøm pore, 4.6 mm ID×25 cm L) in 40 mM aceticacid in methanol/water (58/42, v/v). The component fractions arereferred to specifically as QA-21V1 and QA-21-V2 when describingexperiments or results performed on the further purified components.

In order to purify saponins from Quillaja saponaria Molina bark, aqueousextracts of the Quillaja saponaria Molina bark are dialyzed againstwater. The dialyzed extract is lyophilized to dryness, extracted withmethanol and the methanol-soluble extract is further fractionated bysilica gel chromatography and by reversed-phase high pressure liquidchromatography (RP-HPLC).

TABLE 3 Reversed-phase HPLC peaks designting individual saponins andtheir corresponding retention times. Peak Retention Time (minutes) QA-1solvent front QA-2 4.6 QA-3 5.6 QA-4 6.4 QA-5 7.2 QA-6 9.2 QA-7 9.6 QA-810.6 QA-9 13.0 QA-10 17.2 QA-11 19.0 QA-12 21.2 QA-13 22.6 QA-14 24.0QA-15 25.6 QA-16 28.6 QA-17 35.2 QA-18 38.2 QA-19 43.6 QA-20 47.6 QA-2151.6 QA-22 61.0

As shown above, individual saponins can be separated by reversed-phaseHPLC- At least 22 peaks (designated QA-1 to QA-22) are separable.Individual components are identified by retention time on a VYDAC C4HPLC column as follows in Table 3 herein above. Each peak corresponds toa carbohydrate peak that exhibits only a single band on reversed-phasethin layer chromatography.

The substantially pure QA-7 saponin is characterized as having immuneadjuvant activity, containing about 35% carbohydrate (as assayed byanthrone) per dry weight, having a UV absorption maximum of 205-210 nm,a retention time of approximately 9-10 minutes on RP-HPLC on a VYDAC C4column having 5 μm particle size, 330 Ångstrøm pore, 4.6 mm ID×25 cm Lin a solvent of 40 mM acetic acid in methanol-water (58/42; v/v) at aflow rate of 1 mL/min, eluting with 52-53% methanol from a VYDAC C4column having 5 μm particle size, 330 Ångstrøm pore, 10 mm ID×25 cm L ina solvent of 40 mM acetic acid with gradient elution from 50 to 80%methanol, having a critical micellar concentration of 0.06% (w/v) inwater and 0.07% (w/v) in phosphate buffered saline, causing nodetectable hemolysis of sheep red blood cells at concentrations of 200μg/mL or less, and containing the monosaccharide residues terminalrhamnose, terminal xylose, terminal glucose, terminal galactose,3-xylose, 3,4-rhamnose, 2,3-fucose, 2,3-glucuronic acid, and apiose(linkage not determined).

The substantially pure QA-17 saponin is characterized as having adjuvantactivity, containing about 29% carbohydrate (as assayed by anthrone) perdry weight, having a UV absorption maximum of 205-210 nm, a retentiontime of approximately 35 minutes on RP-HPLC on a VYDAC C4 column having5 μm particle size, 330 Ångstrøm pore, 4.6 mm ID×25 cm L in a solvent of40 mM acetic acid in methanol-water (58/42; v/v) at a flow rate of 1mL/min, eluting with 63-64% methanol from a VYDAC C4 column having 5 μmparticle size, 330 Ångstrøm pore, 10 mm ID×25 cm L in a solvent of 40 mMacetic acid with gradient elution from 50 to 80% methanol, having acritical micellar concentration of 0.06% (w/v) in water and 0.03% (w/v)in phosphate buffered saline, causing hemolysis of sheep red blood cellsat 25 μg/mL or greater, and containing the monosaccharide residuesterminal rhamnose, terminal xylose, 2-fucose, 3-xylose, 3,4-rhamnose,2,3-glucuronic acid, terminal glucose, 2-arabinose, terminal galactoseand apiose (linkage not determined).

The substantially pure QA-18 saponin is characterized as having immuneadjuvant activity, containing about 25-26% carbohydrate (as assayed byanthrone) per dry weight, having a UV absorption maximum of 205-210 nm,a retention time of approximately 38 minutes on RP-HPLC on a VYDAC C4column having 5 μm particle size, 330 Ångstrøm pore, 4.6 mm ID×25 cm Lin a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at aflow rate of 1 mL/min, eluting with 64-45% methanol from a VYDAC C4column having 5 μm particle size, 330 Ångstrøm pore, 10 mm ID×25 cm L ina solvent of 40 mM acetic acid with gradient elution from 50 to 80%methanol, having a critical micellar concentration of 0.04% (w/v) inwater and 0.02% (w/v) in phosphate buffered saline, causing hemolysis ofsheep red blood cells at concentrations of 25 μg/mL or greater, andcontaining the monosaccharides terminal arabinose, terminal apiose,terminal xylose, terminal glucose, terminal galactose, 2-fucose,3-xylose, 3,4-rhamnose, and 2,3-glucuronic acid.

The substantially pure QA-21 saponin is characterized as having immuneadjuvant activity, containing about 22% carbohydrate (as assayed byanthrone) per dry weight, having a UV absorption maximum of 205-210 nm,a retention time of approximately 51 minutes on RP-HPLC on a WDAC C4column having 5 μm particle size, 330 Ångstrøm pore, 4.6 mm ID×25 cm Lin a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at aflow rate of 1 mL/min, eluting with 69 to 70% methanol from a VYDAC C4column having 5 μm particle size, 330 Ångstrøm pore, 10 mm ID×25 cm L ina solvent of 40 mM acetic acid with gradient elution from 50 to 80%methanol, with a critical micellar concentration of about 0.03% (w/v) inwater and 0.02% (w/v) in phosphate buffered saline, and causinghemolysis of sheep red blood cells at concentrations of 26 μg/mL orgreater. The component fractions, substantially pure QA-21-V1 andQA-21-V2 saponins, have the same molecular weight and identical spectraby fast atom bombardment—mass spectroscopy (FAB-MS). They differ only inthat QA-21-V1 has a terminal apiose that is xylose in QA-21-V2 (whichtherefore has two terminal xyloses and no apiose). The two componentsadditionally contain the monosaccharides terminal arabinose, terminalapiose, terminal xylose, 4-rhamnose, terminal galactose, 2-fucose,3-xylose, and 2,3glucuronic acid.

The alkaline hydrolysis products can be prepared as follows. Treatmentof QA-18 by brief alkaline hydrolysis yielded one majorcarbohydrate-containing alkaline hydrolysis product (designatedQA-18-H). Purified QA-18-H was prepared from QA-18 and isolated in thefollowing manner:

One mL QA-18 (5 mg/ml) was incubated with 25 μl 1N NaOH for 15 minutesat room temperature. The reaction was stopped with the addition of 100μl 1N acetic acid. Using these hydrolysis conditions, QA-18 wascompletely converted to a major hydrolysis product (QA-18-H) eluting ina peak with retention time of 8.0 min compared to 66.8 min forunhydrolyzed QA-18, indicating the increased hydrophilicity of QA-18-H.(Chromatography on VYDAC C4 (4.6 mm ID×25 cm L) in 0.1% trifluoroaceticacid in 55/45 methanol/water (v/v) and eluted in a gradient to 64/36methanol/water (v/v) over 180 minutes, flow rate of 1 mL/minute). Thepeak containing pure OA-18-H (retention time 8.0 min) was pooled forfurther characterization. The hydrolysis product of QA-21, designatedQA-21-H, was prepared and purified in the same manner. QA-21-H had aretention time of 9.3 minutes compared to 80.4 minutes for itsunhydrolyzed precursor, QA-21. The hydrolysis products were shown byretention time on HPLC and by reversed-phase thin layer chromatographyto be identical to major hydrolysis products generated using the methodof Higuchi et al., Phytochemistry 26:229 (1987) using mild alkalinehydrolysis in NH₄HCO₃ (Table 4).

TABLE 4 Retention Time of Major Alkaline Hydrolysis Products QA-17-H8.0^(a) QA-18-H 8.0^(a) 8.2^(b) QA-21-H 9.3^(a) 9.5^(b)Hydrolyzed-“Quil-A” 8.2^(a), 9.3^(a) ^(a)Cambridge Biotech hydrolysisconditions: 5 mg/ml saponin, pH 13, reaction time = 15 minutes at roomtemperature. ^(b)Higuchi et al. hydrolysis conditions: 5 mg/ml saponin.6% NH₄HCO₃, methanol/H₂O (1/1, v/v), reaction time = 60 minutes at 100°C.

HPLC Conditions:

VYDAC C4, 5 mm particle size, 330 Ångstrøm pore size, 0.46×25 cm

Solvent A=0.1% trifluoroacetic acid in water

Solvent B—0.1% trifluoroacetic acid in methanol

Gradient=55-64% B/180 minutes

Flow rate—1 ml/min

In addition, these products, QA-18-H and QA-21-H, were shown to be themajor breakdown products from hydrolysis of “Quil-A”, a crude saponinmixture containing QA-7, QA-17, QA-18, and QA-21 as well as othersaponins, indicating that the hydrolysis products QA-21-H and QA-18-Hare the same hydrolysis products isolated by Higuchi et al., supra, forstructural characterization.

Even further preferred saponins according to the present invention arethose described e.g. in EP O436 620 B1, incorporated herein byreference, including fractions termed QHA, QHB, QHC, or similarcompositions of Quillaja saponins.

Acylated quillaja saponins appear to be exceptional since theirmonodesmosidic forms are significantly less effective hemolytic agentsthan their acylated and non-acylated bidesmosidic forms (Pillion, D. J.,et al., J. Pharm. Sci., 84:1276-1279 (1998)).

In addition to Quillaja saponins, saponins originating from Gypsophilaspecies and Saponaria species, including Saponaria officinalis, are alsouseful in accordance with the present invention, particularly Gypsophilaspecies and Saponaria species which have been shown to include“quillajic acid” as the aglycon component of the saponin glycoside.Furthermore, such saponins from Gypsophila species and Saponaria speciespreferably comprise triterpene aglycones with an aldehyde group linkedor attached to position 4, branched oligosaccharides linked by an esterbond in position 28, and a 3-O-glucuronic acid (3-O-glcA) that inQuillaja and Gypsophila is linked to branched oligosaccharides. Saponinsfrom Q. saponaria and S. jenisseenis include acyl moieties, whereassaponin from Gypsophila, Saponaria, and Acanthophyllum do not includeacyl moieties. Each of these non-acylated or deacylated saponins isuseful in the present invention.

Further desirable triterpene saponins are the bidesmosidic saponin,squarroside A, isolated from Acanthophyllum squarrosum; the saponinlucyoside P; and two acylated saponins isolated from Silene jenisseensisWilld. Following is a brief description of these compounds.

Squarroside A is abidesmosidic saponin that contains two oligosaccharidechains linked to C-3 and C-28 of its aglycone gypsogenin. Similar to thegypsophila saponin, it has an aldehyde group linked to C-4 of theaglycone, and a glucuronic acid residue at C-3. In addition, it containsan acetylated fucose residue at C-28. It has been shown that squarrosideA has immunomodulating activity as measured by an in vitrolymphoproliferative test. These apparently nonspecific immunomodulatingeffects were dose-dependent: a suppressive effect at concentrations inthe μg range and a stimulant effect in the pg range.

Lucyoside P is a bidesmosidic saponin that has carbohydrate residuelinked to C-3 and C-28 of its aglycone quillaic acid, and an aldehydegroup at C4. Lucyoside P has a glucuronic acid residue at C-3.

Two acylated saponins have been isolated from the Caryophyllacea Silenejenisseensis. These saponins have carbohydrates linked to C-3 and C28 oftheir agylcone quillaic acid. The carbohydrate residues linked to C-3and C-28 are glucuronic acid and fucose, respectively. The fucoseresidue is acylated with a p-methoxy-cinnamoyl group to yield trans- andcis-p-methoxycinnamoyl tritepene glycosides.

Although the saponins mentioned herein immediately above have analdehyde group, they apparently have no immunostimulating activity or asignificantly reduced immunostimulauing activity, as detected by an invitro chemiluminescence granulocyte assay.

Yet further examples of useful saponins according to the presentinvention pertain to triterpensaponins such as the polar acidicbisdesmosides extracted from e.g. Chikosetsusaponin IV,Calendula-Glycoside C, Chikusetsusaponin V, Achyranthes-Saponin B,Calendula-Glycoside A, Araloside B, Araloside C, Putranjia-Saponin III,Bersamasaponoside, Putranjia-Saponin IV, Trichoside A, Trichoside B,Saponaside A, Trichoside C, Gypsoside, Nutanoside, Dianthoside C,Saponaside D, preferably aescine from Aesculus hippocastanum (T. Pattand W. Winkler: Das therapeutisch wirksame Prinzip der Rosskatanie(Aesculus hippocastanum), Arzneimittelforschung 10(4), 273-275 (1960) orsapoalbin from Gypsophilla struthium (R. Vochten, P. Joos and R.Ruyssen; Physicochemical properties of sapoalbin and their relation tothe foam stability, J. Pharm. Belg. 42, 213-226 (1968).

A number of so-called “modified saponins” obtained from Quillajasaponaria have been disclosed by Kensil et al. in e.g, U.S. Pat. No.5,273,965; U.S. Pat. No. 5,443,829; and U.S. Pat. No. 5,650,398, all ofwhich are incorporated herein by reference. The modified Quillajasaponins typically comprise a methylenealcohol group or a methyleneaminogroup instead of the naturally occurring triterpene aldehyde group. Themodified saponins may be further modified with respect to theirsaccharide moieties.

One interesting saponin composition according to the present inventionis the so-called “7-0-3” composition comprising 7/10 (70%) QH-A, 0/10(0%) QH-B, and 3/10 (30%) QH-C. respectively of each fraction, asdescribed by Ronnberg et al. in Vaccine (1995), vol. 13, no. 14, p.1375-1382, and in Vaccine (1997), vol. 15, no. 17-18, p. 1820-1826.

The ratio between the first saponin and the second saponin in complexesin which both are present are preferably from less than 1000:1 topreferably more than 1:1000. Preferred ratios are about 100:1, forexample about 80:1, such as about 60;1, for example about 50;1, such asabout 40:1, for example about 30:1, such as about 25:1, for exampleabout 20:1, such as about 18:1, for example about 16:1, such as about14:1, for example about 12;1, such as about 10:1, for example about 9:1,such as about 8:1, for example about 7:1, such as about 6:1, for exampleabout 5:1, such as about 4:1, for example about 3:1, such as about 2:1,for example about 1.9:1, such as about 1.8:1, for example about 1.7:1,such as about 1.6:1, for example about 1.5:1, such as about 1.4:1, forexample about 1.3:1, such as about 1.2:1, for example about 1.1:1, suchas about 1:1, for example about 1:1.1, such as about 1:1.2, for exampleabout 1:1.3, such as about 1:1.4, for example about 1:1.5, such as about1:2, for example about 1:3, such as about 1:4, for example about 1:5,such as about 1:10, for example about 1:20, such as about 1:40, forexample about 1:60, such as about 1:80, for example about 1:100.

Sterols and Steroids

Useful sterols are in this context those who bind to saponins formingpart of the complexes according to the invention. Preferred sterols arecholesterols and precursors and derivatives of thereof, as for example,phytosterols, erg. lanosterol, lumisterol, stigmasterol, sitosterol,mycosterols, ergosterol, and thiocholesterol, the last of which can beused for binding a medicament by means of the thiol moiety.Nordihydro-epi-andosterol is a further preferred sterol according to theinvention.

Apart from sterols, the present invention also pertains to complexeswherein at least one first and/or second sterol is substituted partly orwholly by a steroid. In one embodiment, the complexes according to theinvention comprise a steroid compound instead of a sterol compound.Definitions and technical terms used herein to characterize first andsecond sterols apply mutatis mutantis to first and second steroids.

Steroids according to the invention are exemplified herein below in moredetail. As the sterols according to the present invention comprise thecharacteristic skeleton structure of a steroid, the description ofsteroids is also a description of the skeleton of the sterols accordingto the present invention, one of which is cholesterol having CAS(Chemical Abstract) accession no. 57-88-5, or cationic derivativesthereof, in particular cationic derivatives obtained by linking acationic moiety or cationic reactive group to an OH-group, including anOH-group located at position 3 of the steroid skeleton, including theOH-group of cholesterol located at position 3 (C3, or 3-OH).

All steroids are related to a characteristic molecular structurecomposed of 17 carbon atoms arranged in four rings conventionallydenoted by the letters A, B, C, and D and bonded to 28 hydrogen atoms.

This parent structure (1), named gonane and often referred to as thesteroid nucleus, may be modified in a practically unlimited number ofways by removal, replacement, or addition of a few atoms at a time;hundreds of steroids have been isolated from plants and animals, andthousands more have been prepared by chemical treatment of naturalsteroids or by synthesis from simpler compounds

The steroid nucleus is a three-dimensional structure, and atoms orgroups are attached to it by spatially directed bonds. Although manystereoisomers of this nucleus are possible (and may be synthesized), thesaturated nuclear structures of most classes of natural steroids arealike, except at the junction of rings A and B. Simplifiedthree-dimensional diagrams may be used to illustrate stereochemicaldetails. For example, androstane common to a number of natural andsynthetic steroids, exists in two fauns (2 and 3), in which the A/B ringfusions are called cis and trans, respectively.

In the cis isomer, bonds to the methyl group, CH₃, and to the hydrogenatom, H, both project upward from the general plane defined by the restof the molecule, whereas in the trans isomer the methyl group projectsup and the hydrogen projects down. Usually, however, steroid structuresare represented as plane projection diagrams such as 4 and 5, whichcorrespond to 2 and 3, respectively.

The stereochemistry of rings A and B must be specified by showing theorientation of the hydrogen atom attached at C5 (that is, carbon atomnumber 5; steroid numbering is explained below) as either above theplane of the diagram (designated β) or below it (α). The α-, β-symbolismis used in a similar manner to indicate the orientation of anysubstituent group that is attached to a saturated (fully substituted)carbon within the steroid ring system. Groups attached to unsaturatedcarbons lie in the same plane as the adjacent carbons of the ring system(as in ethylene), and no orientation need be specified. When theorientation of a substituent is unknown, it is assigned the symbol ξ.Bonding of β-attached substituents is shown diagrammatically as in 4 bya full line, that of α-substituents by a broken line, as in 5, and thatof ξ-substituents by a wavy line.

Each carbon atom of a steroid molecule is numbered, and the number isreserved to a particular position in the hypothetical parent skeletalstructure (6) whether this position is occupied by a carbon atom or not.

Steroids are named by modification of the names of skeletal rootstructures according to systematic rules agreed upon by theInternational Union of Pure and Applied Chemistry. By attaching prefixesand suffixes to the name of the appropriate root structure, thecharacter of substituent groups or other structural modification isindicated. The prefixes and suffixes include numbers, called locants,indicative of the position in the carbon skeleton at which themodification occurs, and, where necessary, the orientation of asubstituent is shown as α- or β-. The carbon atom at position 3, forexample, is referred to as C3; a hydroxyl group attached to C3 isreferred to as a 3-OH group or, more specifically, as a 3α-OH or 3β-OHgroup. In addition to differences in details of the steroid nucleus, thevarious classes of steroids are distinguished by variations in the sizeand structure of an atomic group (the side chain) attached at position17. The derivations of the names of the more common root structures fromthose of naturally occurring compounds or classes of compounds for whichthey are most typical are known to the skilled artisan. For unambiguoususe of such names, the orientation (α or β) of hydrogen at C5 must bespecified. If no other modification is indicated, the nucleus is assumedto be as shown in (2) and (3), except in the cardanolides andbufanolides: compounds of these types characteristically possess the5β,14β configurations, which, however, are specified.

Preferred second sterols are cationic sterols and sterols comprising atleast one positively charged group at pH=7.0. Preferred sterols compriseor essentially consist of3β-[N-(Dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol)and/or N-(trimethylammonioethane)-carbamoylcholesterol (TC-cholesterol).Further preferred second sterols are described in FIG. 5.

It will be understood that the second sterols comprise cationic sterols,including cationic cholesterols, wherein the OH-group (located atposition 3 in cholesterol) is substituted for a positively chargedgroup, or a group comprising at least one positive charge at pH=7.0.

The ratio between the first sterol and the second sterol in complexes inwhich both are present are preferably from less than 1000:1 topreferably more than 1:1000. Preferred ratios are about 100:1, forexample about 80:1, such as about 60:1, for example about 50:1, such asabout 40:1, for example about 30:1, such as about 25:1, for exampleabout 20:1, such as about 18:1, for example about 16:1, such as about14:1, for example about 12:1, such as about 10:1, for example about 9:1,such as about 8:1, for example about 7:1, such as about 6:1, for exampleabout 5:1, such as about 4:1, for example about 3:1, such as about 2:1,for example about 1.9:1, such as about 1.8:1, for example about 1.7:1,such as about 1.6:1, for example about 1.5:1, such as about 1.4:1, forexample about 1.3:1, such as about 1.2:1, for example about 1.1:1, suchas about 1:1, for example about 1:1.1, such as about 1:1.2, for exampleabout 1:1.3, such as about 1:1.4, for example about 1:1.5, such as about1:2, for example about 1:3, such as about 1:4, for example about 1:5,such as about 1:10, for example about 1:20, such as about 1:40, forexample about 1:60, such as about 1:80, for example about 1:100.

Linker Groups

The deacylsaponins and non-acylsaponins may be directly linked to alipophilic moiety or may be linked via a linking group. By the term“linking group” is intended one or more bifunctional molecules that canbe used to covalently couple the desacylsaponins, non-acylated saponinsor mixtures thereof to the lipophilic molecule.

The linker group in one embodiment covalently attaches to the carboxylicacid group of the 3-O-glucuronic acid moiety on the triterpene corestructure, and to a suitable functional group present on a lipophilicmoiety.

The saponins of the present invention may be directly linked to alipophilic moiety, or a bioactive agent, including a geneticdeterminant, or they may be linked via a linking group. By the term“linker group” is intended one or more bifunctional molecules which canbe used to covalently couple the saponin or saponin mixture to thebioactive agent including a genetic determinant. The linker group may beattached to any part of the saponin.

Typically, the saponins are linked to the lipophilic moiety, or thebioactive agent including a genetic determinant by the preparation of anactive ester of glucuronic acid, a component of the saponins, followedby reaction of the active ester with a nucleophilic functional group onthe bioactive agent including a genetic determinant.

Several lipophile-containing compounds, such as aliphatic amines andalcohols, fatty acids, polyethylene glycols and terpenes, can be addede.g. to the 3-O-glcA residue (3-glucuronic acid residue) ofdeacylsaponins, and to the 3-O-glcA residue of non-acylated saponins.The lipophile may be an aliphatic or cyclic structure that can besaturated or unsaturated. By way of example, fatty acids, terpenoids,aliphatic amines, aliphatic alcohols, aliphatic mercaptans,glycosyl-fatty acids, glycolipids, phospholipids and mono- anddi-acylglycerols can be covalently attached to nonacylated saponins ordesacylsaponins.

Attachment can be via a functional group on a lipophilic moiety thatcovalently reacts with either the acid moiety of the 3-glucuronic acidmoiety, or an activated acid functionality at this position.Alternatively, a bifunctional linker can be employed to conjugate thelipophile to the 3-O-glcA residue of the first and/or second saponin.

Useful fatty acids include C₈-C₂₄ fatty acids, for example C₇-C₂₀ fattyacids, such as C₇-C₁₈ fatty acids. Examples of useful fatty acidsinclude saturated fatty acids such as lauric, myristic, palmitic,stearic, arachidic, behenic, and lignoceric acids: and unsaturated fattyacids, such as palmitoleic, oleic, linoleic, linolenic and arachidonicacids.

Useful aliphatic amines, aliphatic alcohols and aliphatic mercaptansinclude amines and alcohols and mercaptans (R—SH) having astraight-chained or branched, saturated or unsaturated aliphatic grouphaving about 6 to about 24 carbon atoms, for example 6 to 20 carbonatoms, such as 6 to 16 carbon atoms, for example 8 to 12 carbon atoms.Examples of useful aliphatic amines include octylamine, nonylamine,decylamine, dodecylamine, hexadecylamine, sphingosine andphytosphingosine. Examples of useful aliphatic alcohols include octanol,nonanol, decanol, dodecanol, hexadecanol, chimyl alcohol and selachylalcohol.

Useful terpenoids include retinol, retinal, bisabolol, citral,citronellal, citronellol and linalool.

Useful mono- and di-acylglycerols include mono-, and di-esterifiedglycerols,

wherein the acyl groups include from 8 to 20 carbon atoms, including 8to 16 carbon atoms.

Useful polyethylene glycols have the formula H—(O—CH₂—CH₂)_(n)—OH, wheren, the number of ethylene oxide units, is from 4 to 14. Examples ofuseful polyethylene glycols include PEG 200 (n=4), PEG 400 (n=8-9), andPEG 600 (n=12-14).

Useful polyethylene glycol fatty alcohol ethers, wherein the ethyleneoxide units (n) are between 1 to 8, and the alkyl group is from C₈ toC₁₈.

A side-chain with amphipathic characteristics, i.e. asymmetricdistribution of hydrophilic and hydrophobic groups, may facilitate e.g.the accessibility of a triterpene aldehyde to a cellular receptor. It isalso possible that the presence of a negatively-charged carboxyl groupin such a side-chain may contribute to the repulsion of the triterpenegroups, thus allowing them a greater degree of rotational freedom. Thislast factor would most likely increase the accessibility of cellularreceptors to the imine-forming carbonyl group

In one preferred embodiment, when a saponin is linked to a lipophilicmoiety, or to a bioactive agent, including a genetic determinant, bypreparation of an active ester of glucuronic acid, a saponin component,followed by reaction of the active ester with a nucleophilic functionalgroup on the lipophilic moiety, or the bioactive agent including agenetic determinant, such active esters may include the glucuronate ofN-hydroxysuccinimide, sulfo-N-hydroxysuccinimide, hydroxybenzotriazole,and p-nitrophenol. The active esters may be prepared by reaction of thecarboxy group of the saponin with an alcohol in the presence of adehydration agent such as dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI).

The use of EDC to form conjugates is disclosed in U.S. Pat. No.4,526,714 to Feij en et al. and PCT application publication No.WO91/01750, and Arnon, R et al., Pros. Natl. Acad. Sci. (USA)77:6769-6772 (1980), the disclosures of which are fully incorporated byreference herein. The bioactive agent including a genetic determinant isthen mixed with the activated ester in aqueous solution to give theconjugate.

Where a linker group between the saponin and the bioactive agentincluding a genetic determinant is desired, the active ester of thesaponin glucuronate is prepared as described above and reacted with thelinker group, e.g. 2-aminoethanol, an alkylene diamine, an amino acidsuch as glycine, or a carboxy-protected amino acid such as glycinetert-butyl ester.

If the linker contains a protected carboxy group, the protecting groupis removed and the active ester of the linker is prepared (as describedabove). The active ester is then reacted with the bioactive agentincluding a genetic determinant to give the conjugate. Alternatively,the bioactive agent including a genetic determinant may be derivatizedwith succinic anhydride to give an antigensuccinate conjugate which maybe condensed in the presence of EDC or EDCI with a saponin-linkerderivative having a free amino or hydroxyl group on the linker, asdescribed in WO91/01750.

It is also possible to prepare a saponin conjugate comprising a linkerwith a free amino group (derived from an alkylene diamine) and crosslinkthe free amino group with a heterobifunctional cross linker such assulfosuccinimidyl 4-(N-maleimidocyclohexane)-1-carboxylate which willreact with e.g. a free sulfhydryl group of a bioactive agent including agenetic determinant, including any derivative thereof.

The saponin may also be coupled to a linker group by reaction of thealdehyde group of the quillaic acid residue with an amino linker to forman intermediate imine conjugate, followed by reduction with sodiumborohydride or sodium cyanoborohydride. Examples of such linkers includeamino alcohols such as 2-aminoethanol and diamino compounds such asethylenediamine, 1,2-propylenediamine, 1,5-pentanediamine,1,6-hexanediamine, and the like. The bioactive agent including a geneticdeterminant may then be coupled to the linker by first forming thesuccinated derivative with succinic anhydride followed by condensationwith the saponin-linker conjugate with DCC, EDC or EDCI.

In addition, the saponin may be oxidized with periodate and thedialdehyde produced therefrom condensed with an amino alcohol or diaminocompound listed above. The free hydroxyl or amino group on the linkermay then be condensed with the succinate derivative of the bioactiveagent including a genetic determinant in the presence of DCC, EDC orEDCI.

Further useful linker groups are known in the art and examples aredisclosed in e.g. U.S. Pat. No. 6,080,725, which is incorporated hereinby reference.

Contacting Groups

A contacting group according to the present invention is a groupcomprising a lipophilic moiety and a moiety capable of association witha genetic determinant by means of either i) electrostatic interaction,or ii) hydrophobic interaction. Preferably, the contacting groupcomprises a lipophilic moiety and a moiety capable of aligning a stablecomplex with a genetic determinant by means of either i) electrostaticinteraction, or ii) hydrophobic interaction. In one embodiment thecontacting group is capable of association with a genetic determinant bymeans of intercalation.

In one preferred embodiment the genetic determinant is a nucleic acid ora derivative of a nucleic acid, for example the genetic determinant maybe DNA.

The lipophilic moiety may in one embodiment comprise or consist of alipid, however the lipophilic moiety is not limited to compoundscomprising a lipid. For example the lipophilic moiety may be alipophilic peptide or part of a peptide, for example (His)₆.

One preferred contacting group is a cationic compound or groupcomprising at least one positively charged moiety at pH=7.0, which iscapable of association with a genetic determinant, or which is capableof forming a stable complex with a genetic determinant. Such moietiesmay e.g. be found in both saponins and sterols, and they may be found inlipophilic moities according to the present invention.

The overall charge of such a contacting group may be neutral or evenanionic, as long as the contacting group comprises at least onepositively charged moiety at pH=7 capable of association with a geneticdeterminant. Contacting groups can also be neutral in which casepredominantly hydrophobic interactions with a genetic determinant areformed.

Contacting groups according to the present invention does not comprisesuch cationic compounds or such compounds comprising at least onepositively charged moiety at pH=7, which are not capable of associatingwith a genetic determinant. Hence, contacting groups according to theinvention does not include for example phosphatidylcholine,phosphatidylethanolamine, N-decanoyl-N-methylglucami-ne orN-decanoyl-N-methyl-amine.

Examples include, but are not limited to quarternary ammonium compounds;dialkyldimethylammonium compounds; dioctadecyldimethyl ammoniumchloride; dioctadecyldimethyl ammonium bromide;dioctadecyl/octadienyldimethyl ammonium chloride;dioctadecyl/octadienyldimethyl ammonium bromide;dimethyldioctadecylammonium bromide (DDAB), dodecyltrimethylammoniumbromide, hexadecyltrimethylammonium compounds, mixedalkyltrimethylammonium bromide (Cetrimide per BP); andtetradecyltrimethylammonium compounds. Further preferred contactinggroups are illustrated in FIG. 5 (for sterols), and FIGS. 6, 7 and 8(for lipophilic moieties).

Additionally preferred contacting groups includes, but is not limitedto, compounds comprising an essentially planar group that is capable offoaming an intercalation between stacked bases of nucleic acids,including single stranded DNA (ssDNA), double stranded DNA (dsDNA),single stranded RNA (ssRNA), double stranded RNA (dsRNA), RNA and/or DNAcomprising both a single stranded part and a double stranded part, smallnuclear RNA (snRNA), hetroduplexes of RNA and DNA, includinghetroduplexes of RNA and DNA comprising both a single stranded part aswell as a double stranded part, peptide nucleic acids (PNA), lockednucleic acids (LNA), and the like. Examples of such contacting groupsinclude, but is not limited to, acridines and phenanthridines, andderivatives thereof, cumarins, furocumarins, phytoalexins (e.g.psoralens), and derivatives thereof. Such contacting groups may occureither individually in a complex or in any combination with one or moreadditional contacting groups capable of forming an intercalation betweenstacked bases of nucleic acids as described herein above.

Further examples of preferred contacting groups includes, but is notlimited to, indoles and imidazoles, including compounds such e.g.4′,6-diamidio-2-phenylindole, 4′,6-(diimidazolin-2-yl)-2-phenylindole)(obtainable as Hoechst 33258, and Hoechst 33342, respectively),Actinomycin D (such as e.g. 7-Aminoactinomycin D); Cyanine dyes, dimersof cyanine dyes (such as e.g. TOTO®, YOYO®, BOBO™, POPO™) andderivatives thereof.

Any compound having an affinity for a nucleic acid moiety can be used asa contacting group in accordance with the present invention.Accordingly, in addition to intercalating groups, non-intercalatingcontacting groups can also be used. An example is hydroxystilbamidine(Fluoro-Cold™) and derivatives hereof.

A comprehensive list of contacting groups capable of contacting nucleicacids and/or having an affinity to nucleic acids can be found e.g. In“Handbook of Fluorecent Probes and Research Chemicals”, by Richard P.Haugland, Sixth Edition, Molecular Probes (c) 1996, chapter 8, “NucleicAcid Detection”. The compounds listed in the Haugland reference can bereadily modified by the skilled person exerting nothing more thanordinary skill in the art in order to obtain derivatives and analoguesof said compounds listed therein.

Additionally preferred contacting groups are peptides and polypeptidesincluding proteins, enzymes, co-enzymes, antibodies or binding fragmentsof antibodies having an affinity to nucleic acids, including the nucleicacids listed herein immediately above. Examples include, but is notlimited to, nucleic acid binding proteins, including DNA bindingproteins and proteins comprising a helix-turn-helix motif, including analpha-helix-beta-turn-alpha-helix motif associated with the binding ofDNA, Bacteriphage T4 gene 32 protein, E. coli single-stranded bindingprotein, RecA and homologues thereof, including E. coli RecA protein,Cytochrome C, monoclonal antibodies, Fab′ fragments of antibodies, andpolyclonal antibodies.

Contacting groups may also be any nucleic acid capable of forming anassociation with another nucleic acid, and an analogous compound,including PNA and LNA, or a derivative thereof. The association may beformed by hydrogen bonding or any other interaction resulting inbase-pairing and/or duplex formation and/or triplex formation with atleast one genetic determinant. Examples include oligonucleotides andoligonucleotides modified with lipophilic compounds.

Lipophilic Moieties

As described herein above, lipophilic moieties may serve the purpose offacilitating complex formation while at the same time acting as a“docking” group for contacting groups and/or targeting ligands Thelipophilic moieties may thus form part of the complexes according to thepresent invention.

Lipophilic moieties according to the present invention are any moiety,including any residue of a lipophilic molecule, which is attached to orin contact with either i) any suitable functional group of one or morecompounds that is essentially non-polar, or ii) forms an essentiallynon-polar domain within the complexes according to the presentinvention.

The lipophilic moiety can be a portion of an amphipathic compound. Anamphipathic compound is a compound whose molecules contain both polarand non-polar domains. Surfactants are examples of amphipathiccompounds. Surfactants typically possess a non-polar portion that isoften an alkyl, aryl or terpene structure. In addition, a surfactantpossesses a polar portion, that can be anionic, cationic, amphoteric ornon-ionic. Examples of anionic groups are carboxylate, phosphate,sulfonate and sulfate. Examples of cationic domains are amine salts andquaternary ammonium salts. Amphoteric surfactants possess both ananionic and a cationic domain. Non-ionic domains are typicallyderivatives of a fatty acid carboxy group and include saccharide andpolyoxyethylene derivatives.

A lipophilic moiety can also comprise two or more compounds possessingnon-polar domains, wherein each of the compounds has been bonded to alinking group, which, in turn, is covalently attached to a component ofthe complex-according to the present invention, including any saponincomponent and/or any sterol component comprised in said complex,including any residues of said sterol component and any residues of saidsaponin component, including any aglycone part and/or any saccharidepart.

One group of preferred lipophilic moieties are phospholipids such asphosphatidylcholine, phosphatidylethanolamine, triglycerides, fattyacids, and hydrophobic amino acids residues including membrane spanninghydrophobic amino acid segments.

When the complexes according to the present invention comprises i) asaponin derived from a Quil A fraction as described by WO 92/06710,which is incorporated herein by reference, ii) cholesterol and iii)phosphatidylcholine or phosphatidylethanolamine, the complex furthercomprises at least one bioactive agent, including a genetic determinant,including a polynucleotide, including any derivative thereof asdescribed herein. In one embodiment, such complexes have a ratio (weightper weight) of i) lipid and cholesterol to ii) saponin, of more than1:2, such as more than 1.5:2, and a lipid concentration of more than 1mg/ml, for example more than 1.2 mg/ml.

Examples of lipophilic moieties capable of being used in connection withthe present invention are lipids other than sterols, for example fats orfat resembling substances such as e.g. triglycerides or mixedtriglycerides containing fatty acids with up to 50 carbon acids such assaturated fatty acids having for example 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and30 carbon atoms e.g. burytic acid, caprole acid, caprylic acid, capticacid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidicacid, behenic acid, lignoceric acid: unsaturated fatty acids with up to30 carbon atoms, such as hexadecene acid, oleic acid, linoleic acid,linolenic acid, arachidonic acid; hydroxy-fatty acids such as9,10-dihydroxy stearic acid; unsaturated hydroxy fatty acids such ascastor oil; branched fatty acids such as glycerol ethers; waxes i.e.esters between higher fatty acids and monohydric alcohols;phospholipides such as derivatives of glycerol phosphates such asderivatives of phosphatidic acids i.e. lecithin, cephalin, inositolphosphatides, spingosine derivatives with 14, 15, 16, 17, 18, 19 and 20carbon atoms; glycolipids; isoprenoids; sulpholipids; and carotenoids.

Additional examples of lipophilic moieties capable of forming part ofthe complexes according to the present invention are cationic lipids. Itwill be understood that cationic lipids according to the definitionapplied herein are lipids carrying a net positive charge at pH 7.0.

Cationic lipids which may be used in the compositions of the presentinvention include, for example, phosphatidyl ethanolamine, phospatidylcholine, glycero-3-ethylphosphatidyl choline and fatty acyl estersthereof, di- and trimethyl ammonium propane, di- and tri-ethylammoniumpropane and fatty acyl esters thereof. A preferred derivative from thisgroup is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimeth-ylammonium chloride(“DOTMA”).

Additionally, a wide array of synthetic cationic lipids can function inthe present invention. These include common natural lipids derivatizedto contain one or more basic functional groups. Examples of lipids whichcan be so modified include, for example, dimethyldioctadecylammoniumbromide, sphingolipids, sphingo-myelin, lysolipids, glycolipids such asganglioside GM1, sulfatides, glycosphingolipids, cholesterol andcholesterol esters and salts, N-succinyldioleoyl-phosphatidylethanolamine, 1,2,-ioleoyl-sn-glycerol,1,3-dipalmitoyl-2-succinylglycerol,1,2-dipalmitoyl-sn-3-succinylglycerol-,1-hexadecyl-2-palmitoylglycerophosphatidyl ethanolamine andpalmitoylhomocystiene.

In one embodiment, the cationic lipid in the composition of the presentinvention is a fluorinated cationic lipid. Any of the cationic lipidsdescribed herein may be fluorinated by replacing at least one hydrogenatom with a fluorine atom

Specially synthesized cationic lipids also function in the presentinvention, including those compounds of formula (I), formula (II) andformula (III), described in U.S. Pat. No. 6,120,751, the disclosure ofwhich is hereby incorporated by reference herein in its entirety.

Further examples of lipophilic moieties capable of forming part ofcomplexes according to the present invention are, for example,N,N′-Bis(dodecyaminocarbonyl-methylene)-N,N′-bis(β-N,N,N-trimethylammoniume-thyl-aminocarbonyl-methylene)-ethylenediaminetetraiodide;N,N″-Bis(hexadecylamino-carbonyl-methylene)-N,N′,N″-tris(β-N,N,N-trimethyla-mmoniumethylaminocarbonyl-methylenediethylene-triaminehexaiodide:N,N′-Bis(dodecylaminocarbonylmethylene)-N,N″-bis(β-N,N,N-trimethyla-mmoniumethylaminocarbonylmethylene)cyclohexylene-1,4-diaminetetraiodide;1,1,7,7-tetra-(β-N,N,N,N-tetramethylammoniumethylamino-carbonylmethylene)-3-hexadecylaminocarbonylmethylene-1,3,7-triaazaheptaneheptaiodide; and N,N,N′N′-tetra(β-N,N,N-trimethylammoniumethylaminocarbonylmethylene)-N′-(1,2-dioleoylglycero-3-phosphoethanolminocarbonylmethylene)-diethylenetriaminetetraiodide.

In one preferred embodiment, the cationic lipid is a fluorinatedcationic lipid. Any of the cationic lipids described herein may befluorinated by replacing at least one hydrogen atom with a fluorineatom. One skilled in the art will recognize that countless other naturaland synthetic variants carrying positive charged moieties will alsofunction in the invention.

In addition to the cationic lipids described above, other suitablelipids which may be used in the present invention include, for example,fatty acids, lysolipids, fluorinated lipids, phosphocholines, such asthose associated with platelet activation factors (PAF) (Avanti PolarLipids, Alabaster, Ala.), including 1-alkyl-2-acetoyl-sn-glycero3-phosphocholines, and 1-alkyl-2-hydroxy-sn-glycero 3-phosphocholines,which target blood clots; phosphatidylcholine with both saturated andunsaturated lipids, including dioleoylphosphatidylcholine;dimyristoylphosphatidylcholine (DMPC);dipentadecanoylphosphatidylcholine-dilauroylphosphatidylcholine;dipalmitoylphosphatldylcholine (DPPC); distearoylphosphatidylcholine(DSPC); and diarachidonylphosphatidylcholine (DAPC);phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine,dimyristoylphosphatidylethanolamine (DMPE),dipalmitoylphosphatidylethanolamine (DPPE) anddistearoylphosphatidylethanolamine (DSPE); phosphatidylserine;phosphatidylglycerols, including distearoylphosphatidylglycerol (DSPG);phosphatidylinositol; sphingolipids such as sphingomyelin; glycolipidssuch as ganglioside GM1 and GM2; glucolipids; sulfatides;glycosphingolipids; phosphatidic acids, such as dipalmitoylphosphatidicacid (DPPA) and distearoylphosphatidic acid (DSPA); paimitic acid;steaho acid; arachidonic acid; oleic acid; linolenic acid; linoleicacid; myristic acid; synthetic lipids described in U.S. Pat. No.5,312,617, the disclosure of which is hereby incorporated by referenceherein in its entirety.

Additionally preferred lipophilic moieties include, but is not limitedto glycolipids, phosphatldylethnolamine, phosphatidylcholine,phosphatydllinositol, phosphatidylserine, phosphatidylglycerol,including derivatives thereof. Further preferred lipophilic moieties aresphingomyelin, diphosphatidylglycerol (Cardiolipin), phosphatidic acid,Tfx™ Reagents, including Tfx™-10 Reagent, Tfx™-20 Reagent, and Tfx™-50Reagent, and 1,2-Diacyl-sn-Glycero-3-Ethylphosphocholine compounds,particular compounds wherein the acyl groups, independently fromanother, is selected from the group consisting of lauroyl, myristoyl,palmitoyl, stearoyl, oleoyl, palmitoyl-oleoyl, and of dilauroyl,dimyristoyl, dipaimitoyl, distearoyl, dioleoyl (DOPC+). L-α-DioleoylPhosphatidylethanolamine, or1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE) represents oneparticularly preferred lipophilic moiety.

Additionally preferred are DOTAP; DDAB (dimethyl dioctadecylammoniumbromide); 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DOPE;L-β,γ-Dioleoyl-α-cephalin; 3-sn-Phosphatidylethanolamine, 1,2-dideoylN-(1-[2,3-Dioleoyloxy]propyl)-N,N,N-trimethylammonium; Dioctadecyldimethyl ammonium bromide; Avridine (CP-20,961), and stearyl tyrosine.

One particularly interesting lipophilic moiety is Monophosphoryl lipid A(MPL) as described by Baldridge and Crane (1999) in Methods, vol. 19,no. 1, p. 103-107; by Zhou and Huang (1993) in Vaccine, vol. 11, no. 11,p. 1139-1144. and by Rudbach et al. (1995) in Chap. 13. in “The Theoryand Practical Application of Adjuvants” (Stewart-Tull, ed), Wiley &Sons, Ltd. Cationic derivatives of MPL are also included in the presentinvention. MPL can be obtained from Corixa Corp. (www.corixa.com).

Additionally preferred lipolytic moieties are lipids bearing polymers,such as chitin, hyaluronic acid, polyvinylpyrrolidone or polyethyleneglycol (PEG), also referred to herein as “pegylated lipids” withpreferred lipid bearing polymers including DPPE-PEG (DPPE-PEG), whichrefers to the lipid DPPE having a PEG polymer attached thereto,including, for example, DPPE-PEG5000, which refers to DPPE havingattached thereto a PEG polymer having a mean average molecular weight ofabout 5000: lipids bearing sulfonated mono-, di-, oligo- orpolysaccharides; cholesterol, cholesterol sulfate and cholesterolhemisuccinate; tocopherol hemisuccinate; lipids with ether andester-linked fatty acids; polymerized lipids (a wide variety of whichare known in the art); diacetyl phosphate; dicetyl phosphate;stearylamine; cardiolipin; phospholipids with short chain fatty acids ofabout 6 to about 8 carbons in length; synthetic phospholipids withasymmetric acyl chains, such as, for example, one acyl chain of about 6carbons and another acyl chain of about 12 carbons; ceramides; non-ionicliposomes including niosomes such as polyoxyalkylene (e.g.,polyoxyethylene) fatty acid esters, polyoxyalkylene (e.g.,polyoxyethylene) fatty alcohols, polyoxyalkylene (e.g., polyoxyethylene)fatty alcohol ethers, polyoxyalkylene (e.g., polyoxyethylene) sorbitanfatty acid esters (such as the class of compounds referred to as TWEEN®,including, for example, TWEEN® 20, TWEEN® 40 and TWEEN® 80, commerciallyavailable from ICI Americas, Inc., Wilmington, Del.), glycerolpolyethylene glycol oxystearate, glycerol polyethylene glycolricinoleate, alkyloxylated (e.g., ethoxylated) soybean sterols,alkyloxylated (e.g., ethoxylated) castor oil,polyoxyethylene-polyoxy-pro-pylene polymers, and polyoxyalkylene (e.g.,polyoxyethylene) fatty acid stearates; sterol aliphatic acid estersincluding cholesterol sulfate, cholesterol butyrate, cholesterolisobutyrate, cholesterol palmitate, cholesterol stearate, lanosterolacetate, ergosterol palmitate, and phytosterol n-butyrate; sterol estersof sugar acids including cholesterol glucuronide, lanosterolglucuronide, 7-dehydrocholesterol glucuronide, ergosterol glucuronide,cholesterol gluconate, lanosterol gluconate, and ergosterol gluconate;esters of sugar acids and alcohols including lauryl glucuronide,stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoylgluconate, and stearoyl gluconate; esters of sugars and aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid and polyuronic acid; saponinsincluding sarsasapogenin, smilagenin, hederagenin, oleanolic acid, anddigitoxigenin; glycerol dilaurate, glycerol trilaurate, glyceroldipalmitate, glycerol and glycerol esters including, glyceroltripalmitate, glycerol distearate, glycerol tristearate, glyceroldimyristate, glycerol trimyristate; long chain alcohols includingn-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, andn-octadecyl alcohol;6-(5cholesten-3β-yloxy)-1-thio-β-D-galacto-pyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)-hexyl-6-amino-6deoxy-1-thio-β-D-galactopyranoside; 6-(5cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-α-D-manno pyranoside;12-(((7′-diethylaminocoumarin-3-yl)-carbonyl)methylamino)-octadecanoicacid;N-[12-(((7′-diethylaminocoumarin-3-yl)-carbonyl)methylamino)-octadecanoyl]-2-aminopalmiticacid; cholesteryl(4′-trimethylammonio)butanoate;N-succinyldioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoylglycero-phospho-ethanolamine andpalmitoylhomocysteine, and/or any combinations thereof.

One skilled in the art could readily determine the charge (e.g.,cationic, anionic or neutral) of any of the lipids described herein. Ina preferred embodiment, the lipids described herein are fluorinatedlipids. As one skilled in the art will recognize, any of the neutrallipids described herein may be modified to cationic lipids or anioniclipids by methods that are well-known to one skilled in the art. Forexample, any modifiable group on a neutral lipid, such as a secondaryamine, an —OH group or an anionic group or cationic group that have azwitterionic charge balance, may be chemically modified to add orsubtract a charge to the neutral lipid.

When a neutral lipid is used in the compositions of the presentinvention, the neutral lipid is preferably a phosphocholine, asphingolipid, a glycolipid, a glycosphingolipid, a phospholipid or apolymerized lipid.

Examples of polymerized lipids include unsaturated lipophilic chainssuch as alkenyl or alkynyl, containing up to about 50 carbon atoms.Further examples are phospholipids such as phosphoglycerides andsphingolipids carrying polymerizable groups; and saturated andunsaturated fatty acid derivatives with hydroxyl groups, such as forexample triglycerides of d-1,2-hydroxyoleic acid, including castor oiland ergot oil. Polymerization may be designed to include hydrophilicsubstituents such as carboxyl or hydroxyl groups, to enhancedispersablilty so that the backbone residue resulting frombiodegradation is water soluble. Suitable polymerizable lipids are alsodescribed, for example, by Klaveness et al, U.S. Pat. No. 5,536,490, thedisclosure of which is hereby incorporated by reference herein in itsentirety.

Even further examples of lipophilic moieties capable of forming part ofcomplexes according to the present invention are those e.g. described inEP 0 109 952 B1, incorporated herein by reference, and in EP 0 436 620B1, incorporated herein by reference. In particular, the lipophilicmoiety may be a phospholipid such as phosphatidyl-ethanolamine andphosphatidylcholine.

The lipophilic moiety may comprise a lipophilic receptor moleculecapable of binding a cell-binding component such as e.g. an antigen.Examples of such receptors are e.g. glycolipids, for example the choleratoxin's receptor ganglioside GM1 and fucosylated blood group antigen.The cell-binding component can then function as a transport molecule.

Lipophilic Moieties Bound to Polymers

In one embodiment, the lipophilic moiety of the complex according to theinvention is covalently bonded to at least one polymer including, forexample, hydrophilic polymers. Suitable hydrophilic polymers forcovalent bonding to lipids include, for example, polyalkyleneoxides suchas, for example, polyethylene glycol (PEG) and polypropylene glycol(PPG), polyvinyl-pyrrolidones, polyvinylalkylethers, such as apolyvinylmethyl ether, polyacrylamides, such as, for example,polymethacrylamides, polydimethyl-acrylamides andpolyhydroxy-propylmethacrylamides, polyhydroxyakyl(meth)-acrylates, suchas polyhydroxyethyl acrylates, polyhydroxypropyl methacrylates,polyalkyloxazolines, such as polymethyloxazolines andpolyethyloxazoliries, polyhydroxyalkyloxazolines, such aspolyhydroxyethyloxazolines, polyhyhydroxypropyloxazolines, polyvinylalcohols, polyphosphazenes, poly(hydroxyalkylcarboxylic acids),polyoxazolidines, polyaspartamide, and polymers of sialic acid(polysialics).

Preferably, the hydrophilic polymers are polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polypropylene glycol, apolyvinylalkylether, a polyacrylamide, a polyalkyloxazoline, apolyhydroxyalkyloxazoline, a polyphosphazene, a polyoxazolidine, apolyaspartamide, a polymer of sialic acid, apolyhydroxyalkyl(meth)acrylate or a poly(hydroxyalkylcarboyxlic acid).

More preferably, the hydrophilic polymers are PEG, PPG,polyvinylalcohol, polyvinylpyrrolidone and copolymers thereof, with PEGand PPG polymers being more preferred and PEG polymers being even morepreferred. The polyethylene glycol may be, for example, PEG 2000, PEG5000 or PEG 8000, which have weight average molecular weights of 2000,5000 and 8000 daltons, respectively.

Preferably, the polyethylene glycol has a molecular weight of about 500to about 20,000, more preferably from about 1,000 to about 10,000. Othersuitable polymers, hydrophilic and otherwise, will be apparent to oneskilled in the art based on the present disclosure.

Exemplary lipids which are covalently bonded to hydrophilic polymersinclude, for example, dipalmitoylphosphatidylethanolamine-PEG,dioleoylphosphatidylethanolamine-PEG anddistearylphosphatidylethanolamine-PEG, more preferablydipalmitoylphosphatidylethanolamine-PEG.

Liposomes

The above-mentioned polymers which may in one embodiment be attached toe.g. a lipophilic moiety of the complexes according to the invention.The attachment may be by means of alkylation or acylation reactions andthis is useful for improving the stability and size of the distributionof liposomes comprising the complexes according to the invention.

The liposomes may be prepared e.g. as described by Lipford and Wagner(1994) in Vaccine, vol. 12, no. 1, p. 73-80, incorporated herein byreference. General liposomal preparatory techniques which may be adaptedfor use in the preparation of liposome compositions pertaining to thepresent invention are discussed, for example, in U.S. Pat. Nos.4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and4,921,706; U.K. Patent Application GB 2193095A; InternationalApplication Serial Nos. PCT/US85/01161 and PCT/US89/05040; Mayer et al.,Biochimica et Blophysica Acta, 858:161-168 (1986); Hope et al.,Biochimica et Biophysica Acta, 812:55-65 (1985); Mayhew et al., Methodsin Enzymology, 149:64-77 (1987); Mayhew et al., Biochimica et BiophysicaActa, 755:169-74 (1984); Cheng et al, Investigative Radiology, 22:47-55(1987); and Liposome Technology, Gregoriadis, G., ed., Vol. 1, pp.29-31, 51-67 and 79-108 (CRC Press Inc., Boca Raton, Fla. 1984), thedisclosures of each of which are hereby incorporated by referenceherein.

Accordingly, the liposome compositions of the invention comprising thecomplexes of the invention may be prepared using any one of a variety ofconventional liposomal preparatory techniques which will be apparent toone skilled in the art, including, for example, solvent dialysis, Frenchpress, extrusion (with or without freeze-thaw), reverse phaseevaporation, simple freeze-thaw, sonication, chelate dialysis,homogenization, solvent infusion, microemulsification, spontaneousformation, solvent vaporization, solvent dialysis, French pressure celltechnique, controlled detergent dialysis, and others, each involving thepreparation of the compositions in various fashions. See, e.g., Maddenet al., Chemistry and Physics of Lipids, 53:37-46 (1990), the disclosureof which is hereby incorporated herein by reference.

Suitable freeze-thaw techniques are described, for example, inInternational Application Serial No. PCT/US89/05040, filed Nov. 8, 1989,the disclosure of which is hereby incorporated herein by reference inits entirety. Methods, which involve freeze-thaw techniques arepreferred in connection with the preparation of liposomes. Preparationof the liposomes may be carried out in a solution, such as an aqueoussaline solution, aqueous phosphate buffer solution, or sterile water.The liposomes may also be prepared by various processes which involveshaking or vortexing, which may be achieved, for example, by the use ofa mechanical shaking device, such as a Wig-L-Bug™ (Crescent Dental,Lyons, Ill.), a Mixomat (Degussa A G Frankfurt, Germany), a Capmix (EspeFabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, Oberay Germany),a Silamat Plus (Vivadent, Lechtenstein), or a Vibros (Quayle Dental,Sussex, England). Conventional microemulsification equipment, such as aMicrofluidizer™ (Microfluidics, Woburn, Mass.) may also be used.

Bioactive Agents

The complexes according to the present invention may further compriseone or more bioactive agents. Examples of bioactive agents aretherapeutic agents, diagnostic agents, targeting ligands, and geneticdeterminants. However, targeting ligands and genetic determinants mayalso be used without being bioactive agents. In the latter case, thetargeting ligand is merely targeting the complex to a desired cellularlocation, or a predetermined region of a patient, and the geneticdeterminant may be e.g. DNA encoding a bioactive agent in the form ofthe corresponding polypeptide. However, the DNA itself may also act as abioactive agent in this respect.

Charged bioactive agents, such as DNA, can be readily incorporated intothe complexes through e.g. covalent or non-ovalent interactions, such asionic or electrostatic interactions, formed between second sterols,second saponins, or, in the absence of the aforementioned, a contactinggroup.

The bioactive agent, e.g. DNA, may be added to the complex at theinitial stage of preparation thereof. However, Also the DNA may also beadded at a later stage when e.g. the components of the complex have beenmixed and complex formation achieved.

A wide variety of bioactive agents may be delivered to a predeterminedcellular location, or to a particular region of a patient, by using thepresent invention based on complexes comprising at least one bioactiveagent. Suitable bioactive agents include, for example, contrast agents,genetic determinants, chemotherapeutics, peptides and nucleic acids,including derivatised nucleic acids such as e.g. LNA (locked nucleicacids) and PNA (peptide nucleic acids).

One preferred bioactive agent is a genetic determinant, which includes,for example, nucleic acids, RNA and DNA, of either natural or syntheticorigin, including recombinant RNA and DNA and antisense RNA and DNA,hammerhead RNA, ribozymes, hammerhead ribozymes, antigene nucleic acids,both single and double stranded RNA and DNA and analogs thereof,ribooligonucleotides, deoxyribooligonucleotides, antisenseribooligonucleotides, and antisense deoxyribooligonucleotides.

The complexes of the present invention are also suitable for theadministration of a wide variety of peptide and non-peptide bioactiveagents. Bioactive agents, such as peptides, can also be incorporatedwhen they are hydrophobic, neutral or charged. In many cases by usingthe appropriate complex composition, an interaction can be formedbetween the complex and a peptide of interest, or any other bioactiveagent.

Some examples of peptides which may be incorporated into thecompositions are interferons and other macrophage activation factors,such as lymphokines, muramyl dipeptide (MDP), γ-interferon, α-interferonand β-interferon, and related antiviral and tumoricidal agents; renininhibitors including new-generation anti-hypertensive agents;cholecystokinins (CCK analogs) such as CCK, ceruletide and eledoisin,and related cardiovascular-targeting agents and CNS-targeting agents;leukotrienes and prostaglandins, such as oxytocin, and relatedanti-inflammatory, oxytocid and abortifacient compounds; erythropoietinand analogs thereof, as well as related haematinics; LHRH analogs, suchas leuprolide, buserelin and nafarelin, and related down-regulators ofpituitary receptors; parathyroid hormone and other growth hormoneanalogs; enzymes, such as Dnase, catalase and alpha-1 antrtrypsin;immunusuppressants such as cyclosporin; GM-CSF and otherimmunomodulators; and insulin.

Non-peptides which may be used in the compositions and methods of thepresent invention include, for example, beta-agonists, such asisoproterenol, albuterol, isoetherine and metoproteronol, and relatedanti-asthmatics; steroids, such as flunisolide, and similaranti-asthmatics; cholinergic agents, such as cromolyn, and relatedanti-asthmatics; and 5-lipoxygenase inhibitors, such as zileuton and thehydrpxyurea compound described above, and related leukotrieneinhibitors.

Bioactive agents that act as antineoplastics and antibiotics may also bedelivered using the compositions of the present invention. Among theseare included, for example, antibiotics such as p-aminosalicylic acid,isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochlorideethionamide, pyrazinamide, rifampin and streptomycin sulfate, dapsone,chloramphenicol, neomycin, ceflacor, cefadroxil, cephalexin, cephadrineerythromycin, clindamycin, lincomycin, amoxicillin, ampicillin,bacampicillin, carbeniciilin, dicioxicillin, cyclacillin, picioxicillin,hetacillin, methicillin, nafcillin, oxacillin, penicillin (G and V),ticarcillin rifampin, tetracycline and amphotericin B; and antitumordrugs such as methotrexate, fluorourcil, addamycin, mitomycin,ansamitomycin, bleomycin, cystiene arabinoside, arabinosyl adenine,mercaptopolylysine, vincristine, busulfan, chlorambucil, azidothymidine,melphalan (e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine,mitotane, procarbazine hydrochloride dactinomycin (actinomycin D),daunorubicin hydrochloride, doxorubicin hydrochloride, taxol, plicamycin(mithramycin), aminoglutethimide, estramustine phosphate sodium,flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate,testolactone, trilostane, amsacrine (m-AMSA), asparaginase, etoposide(VP-16), interferon α-2a, interferon α-2b, teniposide (VM-26),vinblastine sulfate (VLB), vincristine sulfate, hydroxyurea,procarbazine, and dacarbazine; mitotic inhibitors such as the vincaalkaloids, and steroids such as dexamethasone.

Other bioactive agents that may be used in the compositions of thepresent invention include, for example, LHRH analogs, 5-lipooxygenaseinhibitors, immunosuppressants or bronchodilators; especially preferredmaterials include leuprolide acetate. The LHRHAc-D-2-Nal-D4Cl-Phe-D-3-Pal-Ser-N-MeTyr-D-Lys(Nic)-Leu-Lys(N-Isp)-Pro-D-Ala-NH₂(hereinafter “D-2-Nal”), the 5-lipoxygenase inhibitorN-[3-[5-(4-fluorophenylmethyl)-2-thienyl]-1-methyl-2-propynyl]-N-hydroxyurea, the immunosuppressant cyclosporin A, and the adrenergicbronchodilators isoproterenol arid albuterol. (As used herein, the terms“5-lipoxygenase inhibitor” or “5-LO inhibitor” refer to anyphysiologically active compound capable of affecting leukotrienebiosynthesis.)

Yet another example of bioactive agents capable of being incorporatedinto or associated with the complexes of the present invention includes,but is not limited to, apoptosis inducing proteins Apoptosis inducingproteins includes, but are not limited to BAX, BAD, bcl-xs, p53, andcaspases (Sasaki et al. (2001), Nat. Biotechnol 19, 543-547).

Furthermore, immunemodulators are yet another group of bioactive agentsaccording to the present invention. Examples of immunemodulatorsincludes, but are not limited to, IL-2, IL-4, IL-5, IL-10, IL-12, IL-15,INF-γ, TNF-β

Antigen presenting proteins constitute yet another group of bioactiveagents. Examples of antigen presenting proteins includes MHC class I andMHC class II molecules or fragments thereof capable of antigenpresentation. Preferred MHC class 1 molecules are HLA-B7 and HLA-A2.

A still further example of bioactive agents are cellular toxins such asbacterial toxins including, but not limited to, tetanus toxin andcholera toxin, or fragments thereof having domains capable ofADP-rbosylation (Mowat et al. (2001); J. Immunol. 167, 3398-3405).

Bioactive agents can also be those polypeptides involved inantigen/epitope presentation. Examples include tapasin involved in theloading of MHC class 1 molecule peptide loading, molecular chaperones,heat shock proteins (such as HSP blonging to the conserved Hsp 70, Hsp90, Hsp 110 families (see e.g. Rafiee et al. (2001) Cancer Gene Ther. 8,974-981))

For lipophilic drugs, the interaction may be hydrophobic or van derWaals forces. For charged drugs and lipid head groups, the interactionmay be electrostatic interactions.

Targeting Ligands

In one embodiment the complexes according to the present inventionfurther comprise a targeting ligand for targeting the complex to aparticular location. Such locations may be a particular tissue or thesurface of a particular cell type. Such tissues or cell types typicallyhave a receptor moiety which has an affinity for the targeting ligand ofthe complex.

Suitable targeting ligands according to the present invention are e.g.hydrophobic receptor binding molecules, immunogenic and/or antigenicsubstances. Suitable cell surface receptors capable of contacting orinteracting with the targeting ligands according to the invention aree.g. receptors comprising a lipophilic domaine, or receptors comprisingor essentially consisting of hydrophobic proteins. By using targetingligands in combination with the complexes according to the presentinvention, a greater proportion of complexes reach a predetermineddestination. It is advantageous to use a targeting ligand e.g. when itis desirable to target the complex to a particular mucous membrane knownto contain a component with affinity for a targeting forming part of acomplex according to the invention. A special advantage in this respectis that lipid-containing cell surface receptor binding moieties can beused as an integrated lipophilic moiety in the complexes and optionallyalso replace lipophilic moieties that are used to build up the complex.

Among the receptor-binding components that are comprised by theinvention are, for example, bacterial toxins and their active bindingparts in the form of subunits or fragments or various modifications orderivatives thereof, bacterial fimbriae or other adhesion molecules andtheir active binding parts and/or derivative structures.

Preferred targeting ligands are associated with the complexes covalentlyor non-covalently. The targeting ligand may be bound, for example, via acovalent or non-covalent bond, to at least one of the lipids in thecomposition. Preferably, the targeting ligand is bound to the complexescovalently. In the case of lipid complexes which comprise cholesterol,the targeting ligand is preferably bound to the cholesterolsubstantially only non-covalently, and/or the targeting ligand is boundcovalently to a component of the composition, for example, anotherlipid, such as a phospholipid, other than the cholesterol.

The targeting ligands which are incorporated in the complexes of thepresent invention are preferably substances which are capable oftargeting receptors and/or tissues in vivo and/or in vitro. With respectto the targeting of tissue, the targeting ligands are desirably capableof targeting heart tissue and membranous tissues, including endothelialand epithelial cells. In the case of receptors, the targeting ligandsare desirably capable of targeting GPllbllla receptors or lymphocytereceptors, such as T-cells, B-cells or interleukin-2 receptors.Preferred targeting ligands for use in targeting tissues and/orreceptors, including the tissues and receptors exemplified above, areselected from the group consisting of proteins, including antibodies,antibody fragments, hormones, hormone analogues, glycoproteins andlectins, peptides, polypeptides, amino acids, sugars, such assaccharides, including monosaccharides and polysaccharides, andcarbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors,and genetic determinants, including nucleosides, nucleotides, nucleotideacid constructs and polynucleotides, with peptides being particularlypreferred.

An example of a protein which may be preferred for use as a targetingligand is Protein A, which is protein that is produced by most strainsof Staphylococcus aureus. Protein A is commercially available, forexample, from Sigma Chemical Co. (St. Louis, Mo.). Protein A may then beused for binding a variety of IgG antibodies. Generally speaking,peptides which are particularly useful as targeting ligands includenatural, modified natural, or synthetic peptides that incorporateadditional modes of resistance to degradation by vascularly circulatingesterases, amidases, or peptidases. A useful method of stabilization ofpeptide moieties incorporates the use of cyclization techniques. As anexample, the end-to-end cyclization whereby the carboxy terminus iscovalently linked to the amine terminus via an amide bond may be usefulto inhibit peptide degradation and increase circulating half-life.Additionally, a side chain-to-side chain cyclization or end-to-sidechain cyclization is also useful in inducing stability. In addition, thesubstitution of an L-amino acid for a D-amino acid in a strategic regionof the peptide may offer resistance to biological degradation.

In another embodiment, small peptides which bind the interleukin-1(IL-1) receptor may be used. For example, peptides generated by phagedisplay core sequences of QPY have been shown to be essential forpeptide binding, including, for example, AF12198, a 15-mer with a coresequence of WYQJY, where J is azetidine; and IL-1 antagonists with K_(d)10⁻¹⁰ to 10⁻¹² M, such as AcPhe-Glu,Trp-Pro-Gly-Trp-Tyr-Gln-Aze-Tyr-Ala-Leu-Pro-Leu-CONH₂ orAc-Phe-Glu-Trp-Pro-Gly-Trp-Tyr-Gln-Aze-Tyr-Ala-Leu-Pro-Leu-.

Endothelial-leukocyte adhesion molecules (ELAM's) are antigens which areexpressed by endothelial cells under conditions of stress which thenfacilitate the migration of the leukocytes across the endothelium liningthe vasculature into the surrounding tissues. These sameendothelial-leukocyte adhesion molecules may also be advantageouslyexploited as receptors for targeting of vesicles. These endothelial celladhesion molecules belong to a family known as selectins in which theknown members, such as GMP-140, all participate in endothelial-leukocyteadhesion and include ELAM-1, LAM-1 and the granule membrane protein 140(GMP-140) also known as platelet activation-dependent granule-externalmembrane protein (PADGEM), VCAM-1/INCAM-110 (Vascular AdhesionMoleculelinducible Adhesion Molecule) and ICAM-1 (Intercellular AdhesionMolecule).

A wide variety of different targeting ligands can be selected to bind tothe cytoplasmic domains of the ELAM molecules. Targeting ligands in thisregard may include lectins, a wide variety of carbohydrate or sugarmoieties, antibodies, antibody fragments, Fab fragments, such as, forexample, Fab′2, and synthetic peptides, including, for example,Arginine-Glycine-Aspartic Acid (R-G-D) which may be targeted to woundhealing. While many of these materials may be derived from naturalsources, some may be synthesized by molecular biological recombinanttechniques and others may be synthetic in origin. Peptides may beprepared by a variety of techniques known in the art. Targeting ligandsderived or modified from human leukocyte origin, such as CD11 a/CD18,and leukocyte cell surface glycoprotein (LFA-1), may also be used asthese are known to bind to the endothelial cell receptor ICAM-1. Thecytokine inducible member of the immunoglobulin superfamily, VCAM-1,which is mononuclear leukocyte-selective, may also be used as atargeting ligand. VLA-4, derived from human monocytes, may be used totarget VCAM-1. Antibodies and other targeting ligands may be employed totarget endoglin, which is an endothelial cell proliferation marker.Endoglin is upregulated on endothelial cells in miscellaneous solidtumors. A targeting ligand which may be used to target endoglin is theantibody TEC-11. Thorpe et al, Breast Cancer Research and Treatment,36:237-51 (1995).

Endothelial cell activation in the setting of atherosclerosis is used inthis invention to target the complexes to regions of arteriosclerosisincluding, for example, atherosclerotic plaque. One such target that canbe used is the inducible mononuclear leukocyte endothelial adhesionmolecule recognized by Rb1/9 as an ATHERO-ELAM. The monoclonalantibodies, H4/18 and H18/7, may be used to target endothelial cellsurface antigens which are induced by cytokine mediators. As a preferredembodiment, complexes are targeted to atherosclerotic plaque tonon-invasively detect diseased blood vessels before severe damage hasoccurred, for example, prior to stroke or myocardial infarction, so thatappropriate medical or surgical intervention may be implemented.ATHERO-ELAM is a preferred target and ligands, such as antibodies,peptides, or lectins or combinations thereof may be used to target thiscell surface epitope expressed on endothelial cells in the context ofatherosclerosis. Alternatively, lipoproteins or lipoprotein fragmentsderived from low or high density lipoprotein proteins may be used astargeting ligands. Additionally, cholesterol may be used to target theendothelial cells and localize the lipids, vesicles, and the like, toregions of atherosclerotic plaque. In embodiments which involve the useof cholesterol as a targeting ligand, the cholesterol is preferablyunmodified (non-derivatized) with other chemical groups, moieties andligands.

A targeting ligand directed toward thrombotic material in plaque may beused to differentiate between active and inactive regions ofatherosclerotic plaque. Active plaques in the process of generatingthrombi are more dangerous since they may ultimately occlude a vessel orresult in emboli. In this regard, in addition to low molecular weightheparin fragments, other targeting ligands, such as, for example,antifibrin antibody, tissue plasminogen activator (t-PA), anti-thrombinantibody and fibrin antibodies directed to platelet activation factions,may be used to target active plaque with evolving clots. Most preferredtargeting ligands are those which will target a plasma membraneassociated GPllbllla in activated platelets in addition to targetingP-selectin, and an antibody or associated antibody fragment directed toGPllbllla. The present invention is also useful for detecting regions ofacute myocardial infarction. By attaching anti-myosin (particularlycardiomyosin) antibody or anti-actin antibodies to the lipids, infarctedmyocardium may be detected by the methods of the present invention. Fortargeting to granulation tissue (healing wounds), many of the abovetargeting ligands may be useful, The wound healing tripeptide,arginine-glycine-aspartic acid (RGD), may also be used is a targetingligand in this regard.

As with the endothelial cells discussed above, a wide variety ofpeptides, proteins and antibodies may be employed as targeting ligandsfor targeting epithelial cells. Preferably, a peptide, includingsynthetic, semi-synthetic or naturally-occurring peptides, with highaffinity to the epithelial cell target receptor may be selected, withsynthetic peptides being more preferred. In connection with thesepreferred embodiments, peptides having from about 5 to about 15 aminoacid residues are preferred. Antibodies may be used as whole antibody orantibody fragments, for example, Fab or Fab′₂, either of natural orrecombinant origin. The antibodies of natural origin may be of animal orhuman origin, or may be chimeric (mouse/human). Human recombinant orchimeric antibodies are preferred and fragments are preferred to wholeantibody.

Examples of monoclonal antibodies which may be employed as targetingligands in the present complexes include CALAM 27, which is formed byimmunizing BALEI/c mice with whole human squamous cell carcinoma of thetongue and forming hybridomtas by crossing extracted spleen cells withthose of an NS1 syngeneic myeloma cell line. Gioanni et al, CancerResearch, 47: 4417-4424 (1987). CALAM 27 is directed to surface epitopesof both normal and malignant epithelial cells. Normal lymph nodesgenerally do not contain cells expressing these epitopes. See CancerResearch, 47:4417-4424 (1987). Accordingly, complexes comprising thisantibody can be used to target metastases in the lymph nodes. Themonoclonal antibody 3C2 may be employed as a targeting ligarld fortargeting malignant epithelial cells of serious ovarian carcinoma andendometrioid carcinoma. Another exemplary targeting ligand is Mab 4C7(see Cancer Research, 45:2358-2362 (1985)), which may be used to targetmucinous carcinoma, endometriod carcinoma and mesonephroid carcinoma.For targeting squamous cell carcinoma in head and neck cancer, Mab E48(Biological Abstract, Vol. 099 Issue. 066 Ref 082748) may be used as atargeting ligand. For targeting malignant melanoma, the monoclonalantibody 225.28s (Pathol. Biol., 38 (8):866-869 (1990)) may be employed.The monoclonal antibody mAb2E₁, which is targeted to EPR-1 (effectorcell protease 1), may also be used.

There are a variety of cell surface epitopes on epithelial cells forwhich targeting ligands may be selected. For example, the protein humanpapilloma virus (HPV) has been associated with benign and malignantepithelial proliferations in skin and mucosa. Two HPV oncogenicproteins, E6 and E7, may be targeted as these may be expressed incertain epithelial derived cancers, such as cervical carcinoma. SeeCurr. Opin. Immunol., 6(5);746-54 (1994). Membrane receptors for peptidegrowth factors (PGF-R), which are involved in cancer cell proliferation,may also be selected as tumor antigens. Anticancer Drugs, 5(4):379-93(1994). Also, epidermal growth factor (EGF) and interleukin-2 (IL-2) maybe targeted with suitable targeting ligands, including peptides, whichbind these receptors. Certain melanoma associated antigens (MAA), suchas epidermal growth factor receptor (EGFR) and adhesion molecules (TumorBiol., 15 (4):188-202 (1994)), which are expressed by malignant melanomacells, can be targeted with the complexes provided herein. The tumorassociated antigen FAB-72 on the surface of carcinoma calls may also beselected as a target.

A wide variety of targeting ligands may be selected for targetingmyocardial cells. Exemplary targeting ligands include, for example,anticardiomyosin antibody, which may comprise polyclonal antibody, Fab′₂fragments, or be of human origin, anima origin, for example, mouseorigin, or of chimeric origin.

Additional targeting ligands include dipyridamole; digitalis;nifedipine; apolipoprotein; low density lipoproteins (LDL), includingα-LDL, vLDL and methyl LDL; ryanodine; endothelin; complement receptortype 1; IgG Fc; beta 1-adrenergic; dihydropyridine; adenosine;mineralocorticoid; nicotinic acetylcholine and muscarinic acetylcholine;antibodies to the human alpha 1A-adrenergic receptor; bioactive agents,such as drugs, including the alpha 1-antagonist prazosin; antibodies tothe anti-beta-receptor: drugs which bind to the anti-beta-receptor;anti-cardiac RyR antibodies; endothelin-1, which is an endothelialcell-derived vasoconstrictor peptide that exerts a potent positiveinotropic effect on cardiac tissue (endothelin-1 binds to cardiacsarcolemmal vesicles); monoclonal antibodies which may be generated tothe T-cell receptor α-β receptor and thereby employed to generatetargeting ligands; the complement inhibitor sCR1; drugs, peptides orantibodies which are generated to the dihydropyridine receptor;monoclonal antibodies directed towards the anti-interleukin-2 receptormay be used as targeting ligands to direct the present complexes toareas of myocardial tissue which express this receptor and which may beup-regulated in conditions of inflammation; cyclosporine for directingsimilarly the complexes to areas of inflamed myocardial tissue;methylisobutyl isonitrile; lectins which bind to specific sugars onmembranes of cardiac myocytes and cardiac endothelial cells;adrenomedullin (ADM), which is an endogenous hypotensive andvasorelaxing peptide; atrial natriuretic peptide (ANP); C-typenatriuretic peptide (CNP), which is a 22 amino acid peptide ofendothelial cell origin and is structurally related to atrialnatriuretic peptide but genetically distinct, and possesses vasoactiveand antimitogenic activity; vasonatrin peptide (VNP) which is a chimeraof atrial natriuretic peptide (ANP) and C-type natriuretic peptide (GNP)and comprises 27 amino acids; thrombin; endothelium-derived relaxingfactor (EDRF); neutral endopeptidase 1 (NEP-1); competitive inhibitorsto EDRF, including, for example, NG-monomethyl-L-arginine (L-NMMA);potassium channel antagonists, such as charybdotoxin and glibenclamide;antiheart antibodies, which may be identified in patients withidiopathic dilated cardiomyopathy but which preferably do not elicitcytolysis in the myocardium; antibodies directed against the adeninenucleotide translocator, the branched-chain keto acid dehydrogenase orcardiac myosin; specific antagonists for the endothelin-A receptor,which may be referred to as BQ-123; and antibodies to the angiotensin IIreceptor.

In one interesting embodiment the present invention relates to targetingligands capable of targeting the dendritic cell receptor forendocytosis, DEC-205, as described by Mahnke and Guo (2000) in J. Cell.Biol., vol. 151, no. 3, p. 673-684; and by Jiang and Swiggard (1995) inNature, vol. 375, no. 6527, p. 151-155.

In another interesting embodiment, the present invention relates totargeting ligands in the form of two of the major antigens of heartsarcolemmal are calcium binding glycoproteins which co-purify with thedihydropyridine receptor. Antisera may be raised, including polyclonalor monoclonal antibodies, against purified sarcolemma, and theseantibodies may also be employed as targeting ligands. Purified fractionsof the calcium binding glycoproteins may be isolated from the plasmamembranes of the sarcolemma and then used to generate antibodies. ANP,which, as noted above, may be used as a targeting ligand, can beobtained from cultures of human aortic endothelial cells. ANP isgenerally localized in endothelium, but also may localize to theendothelial or myocardial tissue. ANP may be prepared, for example,using recombinant techniques, as well as by synthesis of the peptideusing peptide synthesis techniques well known to one skilled in the art.It is also possible to use an antibody, either polyclonal or monoclonal,directed towards ANP. Similarly, a peptide directed to ANP may be usedfor targeting endothelial and/or myocardial cells. Both the β and αforms of atrial natriuretic factor may be used as targeting ligands fordirecting the present complexes to myocardial tissue.

Genetic Determinants

Genetic determinants represent one group of bioactive agents accordingto the definition thereof provided herein above. In certain embodimentsof the invention genetic determinants may be present in complex with alipophilic moiety or covalently coupled to a lipophilic moiety.

In aspects of the present invention that relate to gene therapy, thenucleic acid compositions contain either compensating genes or genesthat encode therapeutic proteins. Examples of compensating genes includea gene which encodes dystrophin or a functional fragment, a gene tocompensate for the defective gene in patients suffering from cysticfibrosis, a gene to compensate for the defective gene in patientssuffering from ADA, and a gene encoding Factor VIII. Examples of genesencoding therapeutic proteins include genes which encodeserythropoietin, interferon, LDL receptor, GM-CSF, IL-2, IL-4 and TNF.Additionally, nucleic acid compositions which encode single chainantibody components which specifically bind to toxic substances can beadministered. In some preferred embodiments, the dystrophin gene isprovided as part of a mini-gene and used to treat individuals sufferingfrom muscular dystrophy. In other preferred embodiments, a mini-genewhich contains coding sequence for a partial dystrophin protein isprovided. Dystrophin abnormalities are responsible for both the milderBeckers Muscular Dystrophy (BMD) and the severe Duchenne's MuscularDystrophy (DMD). In BMD dystrophin is made, but it is abnormal in eithersize and/or amount. The patient is mild to moderately weak. In DMD noprotein is made and the patient is wheelchair-bound by age 13 andusually dies by age 20. In some patients, particularly those sufferingfrom BMD, partial dystrophin protein produced by expression of amini-gene delivered according to the present invention can provideimproved muscle function.

In some preferred embodiments, genes encoding IL-2, IL-4, interferon orTNF are delivered to tumor cells which are either present or removed andthen reintroduced into an individual. In some embodiments, a geneencoding γ-interferon is administered to an individual suffering frommultiple sclerosis.

Genetic determinants according to the invention may also be nucleicacids encoding part of or all of an apoptosis inducing protein.Apoptosis inducing proteins includes, but are not limited to BAX, BAD,bcl-xs, p53, and caspases (Sasaki et al. (2001), Nat. Biotechnol. 19,543-547).

Furthermore, genetic determinants according to the invention may also benucleic acids encoding part of or all of a protein, which may functionas an immunemodulator. Examples of immunemodulators includes but are notlimited to IL-2, IL-4, IL-5, IL-10, IL-12, IL15, INF-γ, TNF-β

In addition genetic determinants may also be nucleic acids encoding partof or all of an antigen presenting protein. Examples of antigenpresenting proteins includes MHC class I and MHC class II molecules orfragments thereof capable of antigen presentation. Preferred MHC class Imolecules are HLA-B7 and HLA-A2.

In one embodiment of the invention the genetic determinant may be anantisense nucleic acid (also designated antisense molecule) or aribozyme.

Antisense molecules and ribozymes may also be delivered to the cells ofan individual by introducing a nucleic acid composition which acts as atemplate for copies of such active agents. These agents inactivate orotherwise interfere with the expression of genes that encode proteinswhose presence is undesirable. Nucleic acid compositions which containsequences that encode antisense molecules can be used to inhibit orprevent production of proteins within cells. Thus, production ofproteins such as oncogene products can be eliminated or reduced.Similarly, ribozymes can disrupt gene expression by selectivelydestroying messenger RNA before it is translated into protein. In someembodiments, cells are treated according to the invention using nucleicacid compositions that encode antisense or ribozymes as part of atherapeutic regimen which involves administration of other therapeuticsand procedures. Nucleic acid compositions encoding antisense moleculesand ribozymes use similar vectors as those which are used when proteinproduction is desired except that the coding sequence does not contain astart codon to initiate translation of RNA into protein.

Ribozymes are catalytic RNAs which are capable of self-cleavage orcleavage of another RNA molecule. Several different types of ribozymes,such as hammerhead, hairpin, Tetrahymena group I intron, ahead, andRNase P are known in the art; see S. Edgington, Biotechnology (1992) 10,256-262. Hammerhead ribozymes have a catalytic site which has beenmapped to a core of less than 40 nucleotides Several ribozymes in plantviroids and satellite RNAs share a common secondary structure andcertain conserved nucleotides. Although these ribozymes naturally serveas their own substrate, the enzyme domain can be targeted to another RNAsubstrate through base-pairing with sequences flanking the conservedcleavage site. This ability to custom design ribozymes has allowed themto be used for sequence-specific RNA cleavage; see G. Paolella et al.,EMBO (1992), 1913-1919.) It will therefore be within the skill of one inthe art to use different catalytic sequences from various types ofribozymes, such as the hammerhead catalytic sequence, and design them inthe manner disclosed herein. Ribozymes can be designed against a varietyof targets including pathogen nucleotide sequences and oncogenicsequences. Preferred embodiments include sufficient complementarity tospecifically target the abl-bcr fusion transcript while maintainingefficiency of the cleavage reaction.

As an alternative to targeted antisense delivery, targeted ribozymes maybe used.

The term “ribozyme” refers to an RNA-based enzyme capable of targetingand cleaving particular base sequences in DNA and RNA. Ribozymes caneither be targeted directly to cells, in the form of RNAoligonucleotides incorporating ribozyme sequences, or introduced intothe cell as DNA encoding the desired ribozymal RNA. Ribozymes may beused and applied in much the same way as described for antisense nucleicacids.

Immunogenic Determinants and Antigenic Determinants

Immunogenic determinants and antigenic determinants represent bioactiveagents according to the definition thereof provided herein above. Incertain embodiments of the invention such determinants may be present incomplex with a lipophilic moiety or covalently coupled to a lipophilicmoiety.

The immune system may exhibit both specific and nonspecific immunity(Klein, J., et al., Immunology (2nd), Blackwell Science Inc., Boston(1997)). Generally, B and T lymphocytes, which display specificreceptors on their cell surface for a given antigen, produce specificimmunity. The immune system may respond to different antigens in twoways: 1) humoral-mediated immunity, which includes B cell stimulationand production of antibodies or immunoglobulins [other cells are alsoinvolved in the generation of an antibody response, e.g.antigen-presenting cells (APCs; including macrophages), and helper Tcells (Th1 and Th2)], and 2) cell-mediated immunity (CMI), whichgenerally involves T cells including cytotoxic T lymphocytes (CTLs),although other cells are also involved in the generation of a CTLresponse (e.g., Th1 and/or Th2 cells and APCs).

Nonspecific immunity encompasses various cells and mechanisms such asphagocytosis (the engulfing of foreign particles or antigens) bymacrophages or granulocytes, and natural killer (NK) cell activity,among others.

Nonspecific immunity relies on mechanisms less evolutionarily advanced(e.g., phagocytosis, which is an important host defense mechanism) anddoes not display the acquired nature of specificity and memory,hallmarks of a specific immune response. Nonspecific immunity is moreinnate to vertebrate systems. In addition, cells involved in nonspecificimmunity interact in important ways with B and T cells to produce animmune response.

The key differences between specific and nonspecific immunity are basedupon B and T cell specificity. These cells predominantly acquire theirresponsiveness after activation with a specific antigen and havemechanisms to display memory in the event of future exposure to thatspecific antigen. As a result, vaccination (involving specificity andmemory) is an effective protocol to protect against harmful pathogens.

As stated herein above, immunogenic determinants denote any substancecapable of raising an immune response, including a specific antibodyresponse. Any antigenic determinant is thus also an immunogenicdeterminant, but not all immunogenic determinants are antigenicdeterminants within the meaning of these terms as used herein.

In one embodiment of the invention an immunogenic determinant may be agenetic determinant encoding a substance capable of raising an immuneresponse. Hence the immunogenic determinant may be a nucleic acid, suchas DNA or RNA encoding a (poly)peptide, which in itself is animmunogenic determinant.

The complexes according to the invention may be used as carriers ofbacterial immunogenic determinants and bacterial antigenic determinantsfrom one or more bacteria. Such carriers may be employed as vaccines.Vaccines as used herein shall denote an immunogenic composition capableof raising a protective immune response. When it is desired to use forcomplexes according to the present invention as carriers of immunogenicdeterminants and/or antigenic determinants, the determinant in questionis synthesized in vitro or isolated from a bacteria against which it isdesirable to raise a protective immune response.

Bacteria for which vaccines can be formulated in accordance with thepresent invention include, but are not limited to: Helicobacter pylori,Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum,Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus,Streptococcus spp., Streptococcus pyogenes, Streptococcus pneumoniae,Streptococcus viridans, Enterococcus faecalis, Neisseria menirigitidis,Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonellatyphi, Vibrio cholera, Pasteurella pestis, Pseudomonas aeruginosa,Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridiumdifficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponemaspp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilusducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetellaparapertussis, Bordetella bronchiseptica, hemophilus influenza,Escherichia coli, Shigella spp., Erlichia spp., and Rickettsia spp.

Bacterial immunogenic determinants and antigenic determinants can benative, recombinant or synthetic. Such bacterial immunogenicdeterminants and/or antigenic determinants include, but are not limitedto, selectins or lectins from bacteria that bind to carbohydratedeterminants present on cell surfaces: and bacteria receptors forproteins, such as fibronectin, laminin, and collagens.

Vaccines of the present invention may also include one or moreimmunogenic determinants and/or antigenic determinants from a particularvirus to form a vaccine. Viruses for which vaccines can be formulatedinclude, but are not limited to: Influenza viruses, Parainfluenzaviruses, Mumps virus, Adenoviruses, Respiratory syncytial virus,Epstein-Barr virus, Rhinoviruses, Polioviruses, Coxsackieviruses,Echoviruses, Rubeola virus, Rubella virus, Varicell-zoster virus, Herpesviruses (human and animal), Herpes simplex virus, Parvoviruses (humanand animal), Cytomegalovirus, Hepatitis viruses, Human papillomavirus,Alphaviruses, Flaviviruses, Bunyaviruses, Rabies virus, Arenaviruses,Filoviruses, HIV 1, HIV 2, HTLV-1₁ HTLV-II, FeLV, Bovine LV, FeIV,Canine distemper virus, Canine contagious hepatitis virus, Felinecalicivirus, Feline rhinotracheitis virus, TGE virus (swine), and Footand mouth disease virus.

Viral immunogenic determinants and/or antigenic determinants can benative, recombinant or synthetic. Such viral immunogenic determinantsand/or antigenic determinants include, but are not limited to, viralproteins that are responsible for attachment to cell surface receptorsto initiate the infection process, such as (i) envelope glycoproteins ofretroviruses (HIV, HTLV, FeLV and others) and herpes viruses, and (ii)the neuramidase of influenza viruses. Additionally, peptides derivedfrom such viral proteins can be employed, can be employed, either free,or associated non-covalently, or conjugated covalently to a suitablecarrier.

Vaccines of the present invention may also include one or more tumorassociated immunogenic determinants and/or antigenic determinants. Tumorassociated immunogenic determinants and/or antigenic determinants can benative, recombinant or synthetic. Such tumor associated immunogenicdeterminants and/or antigenic determinants include, but are not limitedto, killed tumor cells and lysates thereof, MAGE-1 or MAGE-3 and peptidefragments thereof, Human chorionic gonadotropin (HCG) and peptidefragments thereof, Carcinoembryonic antigen (CEA) and peptide fragmentsthereof, Alpha fetoprotein (AFP) and peptide fragments thereof,Pancreatic oncofetal antigen and peptide fragments thereof, MUC-1 andpeptide fragments thereof, CA 125, 15-3, 19-9, 549, 195 and peptidefragments thereof, Prostate-specific antigens (PSA) and peptidefragments thereof, Prostaterspecific membrane antigen (PSMA) and peptidefragments thereof, Squamous cell carcinoma antigen (SCCA) and peptidefragments thereof, Ovarian cancer antigen (OCA) and peptide fragmentsthereof, Pancreas cancer associated antigen (PaA) and peptide fragmentsthereof, Her1/neu and peptide fragments thereof, gp-100 and peptidefragments thereof, mutant K-ras proteins and peptide fragments thereof,mutant p53 and peptide fragments thereof, truncated epidermal growthfactor receptor (EGFR), and chimeric protein p210_(BCR-ABL).

Peptides that are derived from these tumor associated immunogenicdeterminants and/or antigenic determinants can be employed, either free,or non-covalently associated, or conjugated covalently to a suitablecarrier. Alternatively, gangliosides can be employed, either free,non-covalently associated or conjugated covalently to a suitablecarrier; or oligosaccharide sequences that are specific or predominantlyfound in cancer cells can be employed either free, non-covalentlyassociated or conjugated covalently to a suitable carrier.

In one particularly preferred embodiment, the present invention relatesto complexes, wherein an antigenic determinant, such as e.g. a generallyimmune stimulating epitope against which it is desirable to raise aprotective immune response, is comprised in the complex in combinationwith a polynucleotide, including DNA, such as e.g. plasmid DNA, encodingthe same antigenic determinant, including an immune stimulating epitope.

In another preferred embodiment, the epitope forming part of the complexis presented as a combination of i) the epitope itself, and ii) anantigenic determinant comprising the epitope.

When the complexes are being used in a vaccine formulation, saidformulation can comprise one or more adjuvants. The term “adjuvant” asused herein is any substance whose admixture with an injectedimmunogenic determinant modifies the immune response. Modification ofthe immune response means augmentation, intensification, or broadeningthe specificity of either or both antibody and cellular immuneresponses. Modification of the immune response can also mean decreasingor suppressing certain antigen-specific immune responses such as theinduction of tolerance.

Such adjuvants may be any compound comprising an adjuvant effect knownto the person skilled in the art. For example such adjuvants could be ofmineral, bacterial, plant, synthetic or host origin or they could be oilin water emulsions.

Adjuvants could be selected from the group consisting of: AIK(SO₄)₂,AlNa(SO₄)₂, AINH₄ (SO₄), silica, alum, Al(OH)₃, Ca₃ (PO₄)₂, kaolin,carbon, aluminum hydroxide, muramyl dipeptides,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred toas nor-MDP),N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine(CGP 1 9835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2%squalene/Tween-80® emulsion, lipopoly-saccharides and its variousderivatives, including lipid A, Freund's Complete Adjuvant (FCA),Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (forexample, poly IC and poly AU acids), wax D from Mycobacterium,tuberculosis, substances found in Corynebacterium parvum, Bordetellapertussis, and members of the genus Brucella, liposomes or other lipidemulsions, Titermax, ISCOMS, Qull A, ALUN (see U.S. Pat. Nos. 58,767 and5,554,372), Lipid A derivatives, choleratoxin derivatives, Heat ShockProteins (HSP, see e.g. Lehner et al. (2000), Eur. J. Immunol. 30,594-603), HSP derivatives, LPS derivatives, synthetic peptide matrixesor GMDP, IL-1, IL-2, IL-5, IL-10, IL-12, IL-15; lipopeptides such ase.g. those disclosed by Bessler et al. 1982 (Hoppe Seylers Z. Physiol.Chem. 363, 767-770) and Bessler et al 1985 (J. Immunol., 135,1900-1905); adjuvant active peptides and biomoleculas, such as e.g.TGF-β and fragments thereof, including fragments disclosed in WO01/72331.

In one embodiment of the present invention the vaccine formulationfurther comprises a carrier. The carrier may be present independently ofan adjuvant. The function of a carrier can for example be to increasethe molecular weight of the immunogenic determinant or the antigenicdeterminant in order to increase the activity or immunogenicity of thedeterminant, to confer stability to the determinant, to increase thebiological activity of the determinant, or to increase its serumhalf-life. The carrier may be any suitable carrier known to the personskilled in the art, for example a protein. A carrier protein could bebut is not limited to keyhole limpet hemocyanin, serum proteins such astransferrin, bovine serum albumin, human serum albumin, thyroglobulin orovalbumin, immunoglobulins, or hormones, such as insulin or palmiticacid. For immunization of humans, the carrier must be a physiologicallyacceptable carrier acceptable to humans and safe. However, tetanustoxoid and/or diptheria toxoid are suitable carriers in one embodimentof the invention. Alternatively, the carrier may be dextrans for examplesepharose. The carrier can be linked to the immunogenic determinant orthe antigenic determinant by means of a labile linker such as e.g. alinker of the type disclosed in WO 97/49425.

The immunogenic determinant and/or the antigenic determinant mayindividually be administered more than once, such as twice, for example3 to 5 times, such as 5 to 10 times, for example 10 to 20 times, such as20 to 50 times. The incubation period between each administration mayvary, but is usually within the range of 1 to 7 days, such as 1 to 4weeks, for example 1 to 6 months, such as 6 to 12 months, for example 1to 5 years, such as 5 to 15 years.

The individual may be any mammal, however preferably the individual is ahuman being.

Functional Homologues

Whenever a polypeptide, protein or nucleic acid is specificallymentioned herein by name, the name is meant to include said polypeptide,protein or nucleic acid per se as well as any functional homologuethereof. Hence, genetic determinants, immunogenic determinant, bioactiveagent, antigenic determinants and medicament may for example be any ofthe specifically mentioned polypeptides, proteins or nucleic acids orany functional homologue thereof.

Functional homologues of polypeptides and proteins according to thepresent invention is meant to comprise any polypeptide sequence(s),which are capable of exerting the same or partly the same function.

Functional homologues according to the present invention comprisepolypeptides with an amino acid sequence, which are sharing at leastsome homology with the predetermined polypeptide sequences as outlinedherein above. For example such polypeptides are at least about 40percent, such as at least about 50 percent homologous, for example atleast about 60 percent homologous, such as at least about 70 percenthomologous, for example at least about 75 percent homologous, such as atleast about 80 percent homologous, for example at least about 85 percenthomologous, such as at least about 90 percent homologous, for example atleast 92 percent homologous, such as at least 94 percent homologous, forexample at least 95 percent homologous, such as at least 96 percenthomologous, for example at least 97 percent homologous, such as at least98 percent homologous, for example at least 99 percent homologous withthe predetermined polypeptide sequences as outlined herein above.

Homology may preferably be calculated by any suitable algorithm or bycomputerised implementations of such algorithms for example CLUSTAL inthe PC/Gene program by Intelligenetics or GAP, BESTFIT, BLAST, FASTA andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG). The homology between amino acid sequences may furthermorebe calculated with the aid of well known matrices such as for exampleany one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85,and BLOSUM 90. Functional homologues according to the present inventionmay comprise more than one such substitution, such as e.g. two aminoacid substitutions, for example three or four amino acid substitutions,such as five or six amino acid substitutions, for example seven or eightamino acid substitutions, such as from 10 to 16 amino acidsubstitutions, for example from 15 to 25 amino acid substitution, suchas from 25 to 30 amino acid substitutions, for example from 30 to 40amino acid substitution, such as from 40 to 50 amino acid substitutions,for example from 50 to 75 amino acid substitution, such as from 75 to100 amino acid substitutions, for example more than 100 amino acidsubstitutions.

The addition or deletion of an amino acid may be an addition or deletionof from 2 to 5 amino acids, such as from 5 to 10 amino acids, forexample from 10 to 20 amino acids, such as from 20 to 50 amino acids.However, additions or deletions of more than 50 amino acids, such asadditions from 50 to 200 amino acids, are also comprised within thepresent invention.

The polypeptides according to the present invention, including anyvariants and functional homologues thereof, may in one embodimentcomprise more than 5 amino acid residues, such as more than 10 aminoacid residues. for example more than 20 amino acid residues, such asmore than 25 amino acid residues, for example more than 50 amino acidresidues, such as more than 75 amino acid residues, for example morethan 100 amino acid residues, such as more than 150 amino acid residues,for example more than 200 amino acid residues.

Homologues of nucleic acid sequences within the scope of the presentinvention are nucleic acid sequences, which encodes an RNA and/or aprotein with similar biological function, and which is either

at least 50% identical, such as at least 60% identical, for example atleast 70% identical, such as at least 75% identical, for example atleast 80% identical, such as at least 85% identical, for example atleast 90% identical, such as at least 95% identical

or able to hybridise to the complementary strand of said nucleic acidsequence under stringent conditions.

Stringent conditions as used herein shall denote stringency as normallyapplied in connection with Southern blotting and hybridisation asdescribed e.g. by Southern E. M., 1975, J. Mol. Biol. 98:503-517. Forsuch purposes it is routine practise to include steps ofprehybridization and hybridization. Such steps are normally performedusing solutions containing 6×SSPE, 5% Denhardt's, 0.5% SDS, 50%formamide, 100 μg/ml denaturated salmon testis DNA (incubation for 18hrs at 42° C.), followed by washings with 2×SSC and 0-5% SDS (at roomtemperature and at 37° C.), and a washing with 0.1×SSC and 0.5% SDS(incubation at 68° C. for 30 min), as described by Sambrook et al.,1989, in “Molecular Cloning/A Laboratory Manual”, Cold Spring Harbor),which is incorporated herein by reference.

Homologous of nucleic acid sequences also encompass nucleic acidsequences which comprise additions and/or deletions. Such additionsand/or deletions may be internal or at the end. Additions and/ordeletions may be of 1-5 nucleotides, such as 5 to 10 nucleotide, forexample 10 to 50 nucleotides, such as 50 to 100 nucleotides, for exampleat least 100 nucleotides.

Methods for Treatment of a Patient

In accordance with the present invention there is also provided a methodof conferring a broad based protective immune response againsthyperproliferating cells that are characteristic of hyperproliferativediseases, as well as a method of treating individuals suffering fromhyperproliferative diseases. As used herein, the term“hyperproliferative diseases” is meant to refer to those diseases anddisorders characterized by hyperproliferation of cells. Examples ofhyperproliferative diseases include all forms of cancer and psoriasis.

It has been discovered that introduction of a nucleic acid compositionthat includes a nucleotide sequence which encodes an immunogenic“hyperproliferating cell”-associated protein into the cells of anindividual, results in the production of those proteins in thevaccinated cells of an individual. As used herein, the term“hyperproliferative-associated protein” is meant to refer to proteinsthat are associated with a hyperproliferative disease. To immunizeagainst hyperproliferative diseases, a nucleic acid composition thatincludes a nucleotide sequence which encodes a protein that isassociated with a hyperproliferative disease is administered to anindividual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein which is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Othertumor-associated proteins can be used as target proteins, such asproteins which are found at higher levels in tumor cells, including theprotein recognized by monoclonal antibody 17-1A and folate bindingproteins.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andbiotechnology, as well as epidemiology, allow for the determination ofprobability and risk assessment for the development of cancer in anindividual. Using genetic screening and/or family health histories, itis possible to predict the probability that a particular individual hasfor developing any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer, or are otherwise in remission,are particularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat such arecurrence. Thus, once it is known that individuals have had a type ofcancer and are at risk of a relapse, they can be immunized in order toprepare their immune systems to combat any future appearance of thecancer.

The present invention also provides a method of treating individualssuffering from hyperproliferative diseases. In such methods, theintroduction of nucleic acid compositions serves as animmunotherapeutic, directing and promoting the immune system of theindividual to combat hyperproliferative cells that produce the targetprotein.

The present invention also provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity, including cell receptors and cells which produce“self”-directed antibodies.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoldosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegeners granulomatosis, Crohn'sdisease and ulcerative colitis. Each of these diseases is characterizedby T cell receptors that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of the T cells would elicit an immuneresponse including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs)which are involved in the disease have been characterized. These TCRsinclude Vβ-3, Vβ14, Vβ-17 and Vβ17. Thus, vaccination with a nucleic addcomposition that encodes at least one of these proteins will elicit animmune response that will target T cells involved in RA. See: Howell, M.D., et al., 1991 Proc. Natl. Acad. Sci. USA 88:10921-10925; Paliard, X.,et al., 1991 Science 253:325-329; Williams, W. V., et al., 1992 J. Clin.Invest. 90:326-333; each of which is incorporated herein by reference.

In MS, several specific variable regions of TCRs which are involved inthe disease have been characterized. These TCRs include Vβ-7 and Vβ-10.Thus, vaccination with a nucleic acid composition that encodes at leastone of these proteins will elicit an immune response that will target Tcells involved in MS. See: Wucherpfennig, K. W., et al., 1990 Science248:1016-1019; Oksenberg, J. R., et al., 1990 Nature 345:344-346; eachof which is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs includeVβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vcα7, Vα-14, Vα-15, Vα-16, Vα-28 andVα-12. Thus, vaccination with a nucleic acid composition that encodes atleast one of these proteins will elicit an immune response that willtarget T cells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Genetic vaccines can be preparedusing this information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies which bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of such antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Genetic vaccines can be prepared using thisinformation.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes nucleic acidcompositions that encode the variable region Of such anti-DNA antibodiesfound in the sera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al. 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci.USA 87-1066, which is incorporated herein by reference.

Medicaments

The complexes according to the present invention are used in onepreferred embodiment for promoting the uptake across mucosal membranesand skin surfaces of any one of a large number of medicaments andpharmaceutically active substances. Preferably, such pharmaceuticallyactive substances are polynucleotides or polypeptides. However,pharmaceutical compositions comprising the complexes according to thepresent invention-can he used to increase the uptake of anypharmaceutically active substance, so long as is molecular weight isless than about 250,000 daltons.

Examples of polypeptides that can be administered together with thepharmaceutical compositions of the present invention include, but arenot limited to, insulin, insulin-like growth factor, growth hormone,parathyroid hormone, renin, prolactin, thyroid stimulating hormone,corticotropin, follicle stimulating hormone, chorionic gonadotropin,luteinizing hormone, luteinizing releasing factor, interferon (alpha,beta, and gamma), lymphokines, interleukin, tumor necrosis factor,antibodies (monoclonal and polyclonal), e.g. IgG, enkephalins (see Su,K. S. E., et al., J. Pharm. Scd. 74:394-98 (1985)), calcitonin(McMartin, C. and Peters, G., Delivery Systems For Peptide Drugs, S. S.Davis et al. (eds.), pp. 249-53, Plenum Press New York (1986)),somatostatin (McMartin, C. and Peters, G., Delivery Systems For PeptideDrugs, Davis, S. S., et al. (eds.), pp. 255-63, Plenum Press New York(1986)), methionyl growth hormone (Moore, J. A. et al., Delivery SystemsFor Peptide Drugs, Davis, S. S., et al. (eds.), pp. 317-329, PlenumPress New York (1986)), oxytocin (Hendricks, C. H. and Pose, S. V., J.A. M. A. 175:384-387 (1961)), vasopressin and desmopressin (Richson, D.W. and Robinson, A. G., Ann Int. Med. 103; 228-239 (1985)), luteinizinghormone releasing hormone (Fink, G. et al., J. Endocr. 63:351-360(1974)), nafarelin acetate (Anik, S. T. et al., J. Pharm. Sci.73:684-685 (1984), secretin (Ohwaki, T. et al., J. Pharm. Sci.74(5):550-552 (May, 1985)), glucagon (Pontiroli, A. E. et al., ActaDiabetol Let. 22:102-110 (1985)), pimolol (Kaila, T. et al., J. OcularPharm. 1:79-83 (1985)), thyrotropin-releasing hormone (Sandow, J. andPetri, W., Trans Nas. System. Med., (Chien, Y. W. ed.) Elsevier SciencePublishers B.B., Amsterdam, pp. 183-199 (1985)).

In addition, the compositions of the present invention can also beemployed to increase the uptake across mucosal membranes and skinsurfaces of enzymes, transferases, hydrolases, isomerases, proteases,ligases and oxidoreductases such as e.g. esterases, phosphatases,glycosidases and peptidases; enzyme inhibitors such as leupeptin,chymostatin and pepslatin and growth factors, such as tumor angiogenesisfactor.

Other suitable pharmaceutically active substances are fat-solublesteroids such as progesterone, estrogens and androgens, as well as thefat soluble vitamins A, D, E and K.

In addition to low and high molecular weight polypeptides, thepharmaceutically active substance can be an anti-inflammatory agent(e.g., indomethacin, flurbiprofen, ketoprofen, ibuprofenphenylbutazone), antibiotics (e.g., beta-lactams, aminoglycosides,macrolides, tetracyclines, pryridonecarboxylic acids, phosphomycin),anti-tumor agents (e.g., adriamycin, cisplatin, bleomycin, mitomycin,fluorouracil, vinblastine, vincristine), amino acids (e.g., ascorbicacid, N-acetyltryptophan), antifungal agents, prostaglandins, vitamins,steroids, vaccine antigens, vaccine adjuvants, and antiviral agents(AZT, DDI, acyclovir, idoxuridine, amantadine, and vidarabine).

The medicaments capable of being administered in combination with thecomplexes according to the present invention may be administered eitherprophylactically, therapeutically, or in connection with a diagnosticmethod carried out on the human or animal body. The complexes may alsocomprise a cosmetic agent used for a cosmetic method of treatment of ahuman or any other animal.

The compositions comprising the complexes according to the invention incombination with a medicament may further comprise a biodegradablemicrosphere encapsulating the complexes and the medicament. Thebiodegradable microsphere may comprise any suitable targeting ligandcapable of guiding or targeting the microsphere to any desired location.

Preferred medicaments for rectal administration include hormones,antibiotics, anaesthetics, analgesics, anti-fungal compounds,bactericides, bacteriostats, anti-protozoan compounds, and anti-viralcompounds.

Examples of medicaments capable of being released from a pharmalogicalcomposition according to the invention and into a human or animal bodyinclude, but are not limited to, antihistamines (e.g., dimenhydrinate,diphenhydramine (50-100 mg), chlorpheniramine and dexchlorpheniraminemaleate), analgesics (e.g., aspirin, codeine, morphine (15-300 mg),dihydromorphone, oxycodone, etc.), anti-inflammatory agents (e.g.,naproxyn, diciofenac, indomethacin, ibuprofen, acetaminophen, aspirin,sulindac), gastro-intestinals. and anti-emetics (e.g., metoclopramide(25-100 mg)), anti-epileptics (e.g., phenyloin, meprobamate andnitrazepam), vasodilators (e.g., nifedipine, papaverine, diltiazem andnicardipine), anti-tussive agents and expectorants (e.g., codeinephosphate), anti-asthmatics (e.g. theophylline), anti-spasmodics (e.g.atropine, scopolamine), hormones (e.g., insulin, heparin), diuretics(e.g., ethacrynic acid, bendroflumethiazide), anti-hypotensives (e.g.,propranolol, clonidine), bronchodilators (e.g., albuterol),anti-inflammatory steroids (e.g., hydrocortisone, triamcinolone,prednisone), antibiotics (e.g., tetracycline), antihemorrhoidals,hypnotics, psychotropics, antidiarrheals, mucolytics, sedatives,decongestants, laxatives, antacids, vitamins, stimulants (includingapetite suppressants such as phenylpropanolamine). The above list is notmeant to be exclusive.

Other types of medicaments include flurazepam, nimetazepam, nitrazepam,perlapine, estazolam, haloxazolam, sodium valproate, sodiumcromoglycate, primidone, alclofenac, perisoxal citrate, clidanac,indomethacin, sulpyrine, flufenamic acid, ketoprofen, sulindac,metiazinic acid, tolmetin sodium, fentiazac, naproxen, fenbufen,protizinic acid, pranoprofen, flurbiprofen, diclofenac sodium, mefenamicacid, ibuprofen, aspirin, dextran sulfate, carindacillin sodium, and thelike.

The medicament may be in the form of a physiologically activepolypeptide, which is selected from the group consisting of insulin,somatostatin, somatostatin derivatives, growth hormone, prolactin,adrenocorticotrophic hormone, melanocyte stimulating hormone,thyrotropin releasing hormone, its salts or its derivatives, thyroidstimulating hormone, luteinizing hormone, follicle stimulating hormone,vasopressin, vasopressin derivatives, oxytocin, carcitonin, parathyroidhormone, glucagon, gastrin, secretin, pancreozymin, cholecystokinin,angiotensin, human placental lactogen, human chorionic gonadotropin,enkephalin, enkephalin derivatives, endorphin, interferon (in one ormore of the forms alpha, beta, and gamma), urokinase, kallikrein,thymopoietin, thymosin, motilin, dynorphin, bombesin, neurotensin,caerulein, bradykinin, substance P, kyotorophin, nerve growth factor,polymyxin B, colistin, gramicidin, bacitracin, bleomycin andneocarzinostatin. Furthermore, the medicament may be a polysaccharide,such as heparin, an antitumor agent such as lentinan, zymosan and PS-K(krestin), an aminoglycoside such as e.g. gentamycin, streptomycin,kanamycin, dibekacin, paromomycin, kanendomycin, lipidomycin,tobramycin, amikacin, fradiomycin and sisomicin, a beta-lactamantibiotic, such as e.g. a penicillin, such as e.g. sulbenicillin,mecillinam, carbenicllin, piperacillin and ticarcillin, thienamycin, andcephalosponns such as cefotiam, cefsulodine, cefmenoxime, cefmetazole,cefazolin, cefotaxime, cefoperazone, ceflizoxime and moxalactam, or anucleic acid drug such as e.g. citicoline and similar antitumor agents,for example cytarabine and 5-FU (5-fluorouracil).

Medicaments suitable for vaginal administration are contraceptives,hormones, antibiotics, anaesthetics, analgesics, contraction-preventers,anti-mycotica, bactericides, bacteriostats, anti-protozoan compounds,anti-viral compounds, and compositions for uterus contraction. Othersuitable medicaments may be dermatological medicaments such asantimycotica, antipruritc compositions, and dermoprotectivecompositions.

Medicaments for administration in the ear (otogenic administration) aree.g. antibiotics, corticosteroids, local anaesthetics, and analgesics.

Medicaments for nasal administration are e.g. haemostatica,anti-allergenic compounds, antihistamines, anticholinergica, adrenergic(detumescent) compounds, and local analgesics.

The medicaments can in principle have either local effects, or systemiceffects. In a preferred embodiment the pharmaceutical compositionaccording to the invention comprises at least one medicament that has alocal effect and essentially does not have any systemic effects.

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention can be appliedto any mucous membrane Including the conjunctiva, nasopharynx,orthopharnyx, vagina, colon, urethra, urinary bladder, lung, large(rectal) and small (enteral) intestine. The compositions of the presentinvention can also be administered transdermally, for example, as partof a patch. Preferably, the compositions of the present invention areadministered to the eye as part of eye drops, nasally as part of anaerosol or buccally as part of a solid wafer.

Accordingly, any state of the art method can be used for delivery to anindividual of the complexes or the pharmaceutical compositionscomprising the complexes according to the invention. Examples include,but are not limited to i) associating the complexes or thepharmaceutical compositions comprising the complexes with a cationicliposome, ii) covalently linking the complexes or the pharmaceuticalcompositions comprising the complexes to a transfection agent, and iii)coating minute gold particles with the complexes or the pharmaceuticalcompositions comprising the complexes and using the coated particles fora bio-ballistic transfer.

In addition, the pharmaceutical compositions of the present inventioncan also be formulated in sustained release compositions. For example,the sterol and/or saponin component of the complex and the drug can becombined with a silicone elastomer that releases the complex and drugover a long period of time. The silicone elastomer can also comprisealbumin. See U.S. Pat. No. 4,985,253, the contents of which are fullyincorporated by reference herein. The release rate of the drug from thesilicone elastomer can be controlled by incorporation of a water solubleor fat soluble mixing agent or cosolvent (e.g., polyethylene glycol 400,polysorbate 80, sodium alginate, L-alanine, sodium chloride;polydimethylsiloxane) into the silicone elastomer. Any other additivecan also be incorporated into the silicone elastomer for the purpose ofaccelerating the release rate.

In addition, the pharmaceutically active substance and the complexesaccording to the invention can be formulated in a controlled releasecomposition comprising a polylactide and/or polyglycolide copolymer, acellulose polymer (methyl-, methylhy-droxyethyl-, hydroxypropyl-,hydroxyethyl-, sodium carboxyethyl-cellulose), polyacrylic acid,polymethylmethacrylate, cross-linked polyacrylic acid,polyvinylpyrrolidone, polyvinylalcohol, polyethylene glycol, agarose ora copolymer of styrene and hydroxyethylmethacylate crosslinked withdivinylazobenzene.

Alternatively, the pharmaceutically active substance and the complexesaccording to the invention can be formulated as part of a DEAE-dextranmicrosphere, a starch microspheres, an albumin microspheres, or anyother microsphere or microcapsule made from any pharmaceuticallyacceptable material.

Likewise the microsphere may be provided with a coating. This coatingmay be of a kind that prevents agglomeration or sticking of themicrospheres or prevents evaporation of the drug and/or a solventcomprising the drug inside the microsphere. The invention also foreseesthe use of coatings providing the microsphere with an affinity forspecific cells or tissues. Such an affinity-coating may be in the formof specific amino-acid sequences or even anti-bodies or parts ofantibodies having an affinity for specific proteins. Thereby thedrug-delivery can be targeted to exactly those cells (e.g. cancer cells,metastases) to which the drug should be administered. Likewise thismakes it possible to use the microspheres for diagnostic use and thedrug could in such cases be substituted by a compound suitable forlabelling the targeted cells.

Agents for encapsulation include but are not limited to coilloids,hydrocolloids such as geoatine, exudates such as gum arabic, tragacanth,gum karya, gum ghatti; extracts from seaweed such as agar, alginate,carrageenan and furcellaran; extracts from plants such as pectin andarabinogalactan; extracts from marine and terrestrial animals such asgelatines and other proteinaceous hydrocolloids; flours from seeds suchas guar, locust bean, soya bean; proteins from seeds such as soya beanproteins; flours from cereals such as starches and microcrystallinecellulose, biosynthetic or fermentation derived hydrocolloids such asdextran, xanthan and curdlan; chemically modified hydrocolloids such ascellulose derivatives, including methyl cellulose and other derivatives,including modified starches and low methoxyl pectin; synthetichydrocolloids such as polyvinylpyrrolidone, carboxyvinyl polymers etc.

According to one embodiment of the invention, the microspheres contain ahydrophobic/aerophilic solid material having a maximum average particlesize not exceeding 10 μm (micrometer) and which can be dispersed inwater in the form of discrete micropartides, wherein the amount of solidactive material in the microencapsulated product is from 22 to 71% byweight.

According to another embodiment of the invention, the microspheres maycomprise a microencapsulated oil or fat product, wherein at least oneoil or fat is dispersed in the matrix material as particles or dropshaving an average diameter of less than or equal to 2 μm (micrometer),the oil or fat containing at least 10% by weight of highly unsaturatedfatty acid, preferably omega-3 and omega-6 fatty acids, the level offree fatty acids being below 5.0% by weight and preferably below about0.5% by weight, and the matrix material consisting of caseinate andoptionally at least one carbohydrate. The oil or fat may be a marineoil, preferably a fish oil, containing at least 30% by weight of omega-3fatty acids. Similarly, the oil or fat may be a vegetable oil,preferably borage oil, and preferably containing at least 20%. by weightof omega-3 and/or omega-6 fatty acids. This oil or fat may be a natural,fermented and/or enzymatically reesterified or chemically modified oilor fat, preferably in an amount of from 10 to 65% by weight; the matrixmaterial comprises from 1 to 100% by weight caseinate and from 0 to 70%by weight of at least one carbohydrate selected from the groupconsisting of glucose syrup, maltodextrin, saccharose, maltose orlactose: from 0 to 10% by weight of at least one antioxidant selectedfrom the group consisting of the vitamin antioxidants a-, ss-, r- and6-tocopherols, ascorbic acid and derivatives thereof, carotenoids, androsemary extract, and from 0 to 35% by weight of a spraying agentselected from the group consisting of corn starch, milk proteins,including casein, caseinate and whey proteins, preboiled or gelatinisedstarch, soy bean protein isolates, lactose, tricalcium phosphate, andcalcium carbonate.

In the case of a water-soluble drug, the microsphere may be prepared asa two-phase system with an inner aqueous phase comprising the drug andoptionally drug-retaining or drug-stabilising compounds. This inneraqueous phase can then be emulsified with an oil-phase comprising apolymer to create a water/oil emulsion.

The polymer to be contained in the oil phase in carrying out themicroencapsulation method is a polymer, which is scarcely soluble orinsoluble in water and is biocompatible. Examples are such biodegradablepolymers as aliphatic polymers (e.g. polylactic acid, polyglycolic acid,polycitric acid, polymalic acid), poly-alpha-cyanoacrylic acid esters,poly-beta-hydroxybutyric acid, polyalkylene oxalate (e.g.polytrimethylene oxalate, polytetramethylene oxalate), polyorthoesters,polyorthocarbonates and other polycarbonates (e.g. polyethylenecarbonate, polyethylenepropylene carbonate), and polyamino acids (e.g.poly-γ-benzyl-L-glutamic acid, poly-L-alaine,poly-gamma-methyl-L-glutamic acid). Other biocompatible high polymersare polystyrene, polyacrylic acid, polymethacrylic acid, acrylicacid-methacrylic acid copolymers, polyamides (nylon), polyethyleneterephthalate (tetron), polyamino acids, silicone polymers, dextranstearate, ethylcellulose, acetyl-cellulose, nitrocellulose,polyurethanes, maleic anhydride-based copolymers, ethylene-vinyl acetatecopolymers, polyvinyl acetate, polyvinyl alcohol, polyacrylamide, etc.These polymers may be homopolymers or copolymers of two or moremonomers, or mixtures of the polymers. They may also be in the saltform.

For the emulsification procedure, a known method of effecting dispersionis used. Said method is, for example, the intermittent shaking method,the mixer method using a propeller-shaped stirrer, a turbine-shapedstirrer or the like, the colloid mill method, the hornogeniser method orthe ultrasonication method.

The thus-prepared W/O emulsion is then emulsified into a W/O/Wtriplicate-phase emulsion and subjected to an in-water drying. Thus,said W/O emulsion is further added to a third aqueous phase to give aW/O/W emulsion and thereafter the solvent in the oil phase is removed togive microspheres.

To the external aqueous phase, there may be added an emulsifying agent.As the emulsifying agent, there may be used any one capable of forminggenerally a stable 0/W emulsion, for example an anionic surfactant (e.g.sodium oleate, sodium stearate, sodium lauryl sulfate), a nonionicsurfactant [e.g. polyoxyethylenesorbitan fatty acid ester (Tween 80,Tween 60, products of Atlas Powder Co., U.S.A.), a polyoxyethylenecastor oil derivative (HCO-60, HCO-50, products of Nikko Chemicals,Japan)), polyvinyl pyrrolidone, polyvinyl alcohol,carboxymethylcellulose, lecithin or gelatin. Such emulsifiers may beused either alone or in any combination.

When the complexes according to the invention and pharmaceuticallyactive substance are formulated in a sustained release composition, thecontent of the pharmaceutical substance can be appropriately controlleddepending upon the dose to be administered and the release rate. Whenthe composition is shaped in matrix type preparation, the content of thepharmaceutical substance can usually be from 5 to 40% by weight and,more preferably, not more than 15% by weight, for example, 9% by weightor less. When administering a peptide hormone, its content should be nomore than about 6 to 10% by weight. Albumin, if employed, is present atnot more than 50% by weight, preferably from about 20 to 30% by weight.The silicone elastomer can be contained in an amount of not less than50% by weight, preferably from 70 to 90% by weight.

The sustained release compositions can be prepared by mixing thecomponents in any optional order. When albumin is added, the drug andalbumin are first combined, preferably in a solid state. Alternatively,an aqueous solution of the pharmaceutical substance and albumin can bemixed and the resulting mixture lyophilized to make a solid mixture.This mixture is then dispersed uniformly with an elastomer base,optionally, with a plasticizer (e.g., dimethylpolysiloxane), adding acuring agent thereto and stirring the resultant mixture. The mixture isthen placed in an appropriate mold and cured at room temperature to givea shaped composition. In the alternative, a core material not containinga pharmaceutical substance can be covered with the compositioncomprising a silicone elastomer containing a pharmaceutical substance,optionally containing albumin, to make a shaped composition. Such corematerial can comprise any non-toxic material. Preferably, such corematerial is an elastic polymer.

The sustained release compositions of the present invention can have anyshape that is suitable for effective contact with mucous membranes inbody cavities. For example, when the pharmaceutically active substanceis administered buccally, the sustained release composition can be inthe form of a wafer. When the pharmaceutically active substance isadministered vaginally, the sustained release composition can be in theform of a ring. When administered ocularly, the sustained releasecomposition can be in the form of thin ocular implants.

The compositions of the present invention can also be formulated as partof a chewing gum comprising the gum from the latex of the sapodilla.Preferably, the chewing gum composition also comprises sweeteners(sugar, aspartame and the like) and flavorants (spearmint, wintergreenor peppermint oil and the like) that mask any unpleasant tasteassociated with the pharmaceutically active substance.

When administered ocularly or nasally, the compositions of the presentinvention can be formulated in an aqueous solution buffered to a pH ofbetween 3.0 and 8.0, most preferably pH 5.0-5.4, by means of apharmaceutically acceptable buffer system. Any pharmaceuticallyacceptable buffering system capable of maintaining the pH in thepreferred ranges can be used in the practice of this invention. Atypical buffer will be, for example, an acetate buffer, a phosphatebuffer, a citrate buffer, a succinate buffer, or the like. Theconcentration of buffer can range from between 0.005 and 0.1 molar, mostpreferably about 0.02 molar.

When the compositions of the present invention are administeredocularly, the composition can comprise a solution containing sodium,potassium, magnesium, calcium, chloride and bicarbonate ions as well asdextrose and glutathione. See, for example, U.S. Pat. Nos. 4,550,022 and4,443,432.

Alternatively, the ocular fluid can comprise an aqueous solutioncontaining sodium chloride, potassium chloride, calcium chloride andN-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid. Sodium hydroxidecan be included to establish a pH value of about 7.25 and magnesiumsulfate can also be included. See UK Patent Application GB 2,064,320.See also U.S. Pat. No. 4,938,970, which discloses irrigation solutionsthat do not cause pain when administered to the eye. According to thispatent, the electrolyte solution comprises 2-10 meq/L of K⁺, 0-3 meq/Lof Ca⁺+, 1-5 meq/L of Mg⁺+ and 110-150 meq/L of Na⁺+, buffered to a pHof 6.85-8.0.

Other materials, such as preservatives, salts to achieve the tonic valueof tissue, or other additives indicated by known nasal or ocularformulation chemistry, can be added to these formulations.

By the term “animal” is intended all animals that might derive a benefitfrom the compositions of this invention. Foremost among such animals arehumans: however, the invention is not intended to be so limited, itbeing within the contemplation of the invention to treat any and allsuch animals that can experience the beneficial effects of the presentinvention.

For nasal administration, the compositions of the invention willpreferably be in a container provided with means for enablingapplication of the contained composition to the nasal mucosa, e.g., witha nasal applicator device. Suitable applicators are known in the art andinclude those adapted for administration of liquid compositions to thenasal mucosa in drop or spray form. Since dosing with polypeptidesshould be as accurately controlled as possible, the use of sprayapplicators for which the administered quantity is susceptible toprecise regulation is generally preferred.

Suitable administrators include e.g., atomizing devices, e.g.,pop-atomizers and aerosol dispensers. In the tatter case, the applicatorwill contain the composition of the present invention together with apropellant medium suitable for use in a nasal applicator. The atomizingdevice will be provided with an appropriate spray adapter allowingdelivery of the contained composition to the nasal mucosa. Such devicesare well known in the art.

The container, e.g. nasal applicator or eye drop bottle, can containsufficient composition for a single nasal or ocular dosing or forseveral sequential dosages, e.g. over a period of days or weeks.

Kit-of-Parts

In many aspects the present invention relates to complexes, wherein saidcomplexes further comprises for example a genetic determinant and/or animmunogenic determinant. However, in one embodiment the presentinvention relates to any of the above described complexes, wherein saidcomplex have been admixed with, but is not complexed with one or moreselected from the group consisting of bioactive agents, immunogenicdeterminants, genetic determinants, enzymes, adjuvants and adjuvantactive peptides and biomolecules, and medicaments.

In another embodiment the present invention relates to a kit-of-parts,wherein said kit-of-parts comprises a complex according to the inventionand one or more selected from the group consisting of bioactive agents,immunogenic determinants, genetic determinants, enzymes, adjuvants andmedicaments.

In one embodiment the present invention relates to a kit-of-partscomprising

a) any of complexes described herein above; and

b) an immunogenic determinant; and/or

c) an antigenic determinant; and

wherein the immunogenic determinant is different from the antigenicdeterminant.

In particular such as kit-of-part may be used for a prime-boostimmunisation. Hence the immunogenic determinant may be administered toan individual in need thereof and after an incubation period theantigenic determinant may be administered to said individual.Alternatively, the antigenic determinant may be administered to anindividual in need thereof and after an incubation period theimmunogenic determinant may be administered to said individual.

It is preferred that the immunogenic determinant comprises or encodes atleast one epitope, which is also comprised within the antigenicdeterminant.

The immunogenic determinant may be any of the immunogenic determinantsdescribed herein above. For example the immunogenic determinant may be agenetic determinant encoding a product capable of raising an immuneresponse. In one preferred embodiment, the immunogenic determinantcomprises or essentially consists of a polynucleotide. Saidpolynucleotide preferably encodes an epitope, which is comprised withinthe antigenic determinant.

The immunogenic determinant may be present isolated or as part of acomplex according to the invention or admixed with a vaccineformulation. Hence, for example a complex within the kit-of-parts maycomprise said immunogenic determinant.

The antigenic determinant may be any of the antigenic determinantdescribed herein above. In one preferred embodiment however, theantigenic determinant comprises or essentially consists of apolypeptide. For example the antigenic determinant may be a lipopeptide,such as any of the lipopeptides described in Nardelli et al. (1994),Vaccine 12, 1335-1339; Tam et al. (1998), Dev. Biol. Stand., 92,109-116, Beekman et al. (1997), J. Pept. Res. 50, 357-364; Beekman et al(1999), Vaccine 17, 2043-2050; Bessler et al. (1997), Behring Inst.Mitt., 390-399; Bessler et al. (1997), Int. J. Immunopharmacol., 19,547-550, Deres et al. (1989), Nature 342, 561-564, Hoffmann et al.(1989), Biol. Cham, Hoppe Seyler, 370, 575-582; Schild et al. (1991),Eur. J. Immunol., 21, 2649-2654; Schlecht et al. (1993),Naturwissenschaften, 80, 9-17. Said polypeptide preferably comprises anepitope, which is also comprised within or encoded by the immunogenicdeterminant.

The antigenic determinant may be present isolated or as part of acomplex according to the invention or mixed into a vaccine formulationHence, for example a complex within the kit-of-parts may comprise saidantigenic determinant.

EXAMPLES

The following examples are merely illustrative of preferred embodimentsof the invention and should not be interpreted in any way that islimiting the invention to what is disclosed in the examples.

Example 1 Incorporation of Low-Molecular Weight Substances into ISCOMs

Immune stimulating complexes (ISCOMs) are cage-like structures ofuniform size with a diameter of approximate 40 nm. In general, the termISCOM is used when antigen is inserted into the structures, whereasISCOM-matrix denotes structures without antigen. Several reportsdescribe the formation of ISCOMs with amphipathic proteins like virusmembrane proteins. But also, non-amphipatic proteins like BSA and OVA aswell as hydrophilic proteins linked to fatty acids have been used. Twocrucial components for the formation of ISCOMs are cholesterol andphospholipid that are solubilized by detergent. Many protein antigenscan be brought into solution by detergents compatible with the processof ISCOM-formation whereas other compounds like lipopeptides requiredifferent solvents. The methods reported here allow the embedment ofsuch lipopeptides into the structures of ISCOMs by employing apre-dissolution step in organic solvent (N,N′-dimethylsuffoxide)followed by stepwise dilution with detergent:water. By the use ofdifferent solvents miscible with water (dimethylformamide, ethanol)other low molecular weight compounds can be inserted into ISCOMs. Thisis demonstrated for the fluorescent dye Dil. The influence of organicsolvents for the formation of intact ISCOM structures was alsoinvestigated. For the three solvents tested (dimethylsulfoxide,dimethylformamide, ethanol) the maximum allowable concentration duringthe creation of ISCOMs was between 15%-25%. All preparations of ISCOMswere done in the presence Mega 10, a detergent with high criticalmicelle concentration. To investigate if Mega 10 remained a constituentpart of ISCOM-matrix after dialysis radiolabeled Mega 10 was addedduring the preparation. Dialysis efficiently removed the detergent andonly a negligible amount remained associated with the ISCOM-matrix.

INTRODUCTION

Immune stimulating complexes or ISCOMs were discovered when the adjuvantQuil A was combined with viral proteins in the presence of lipids. Thesecage-like microstructures are as the name indicates generally employedin immunization experiments, but may also be of use in non-immunologicalapplications due to their characteristic properties ISCOMs are stablerigid structures that can be stored in aqueous solutions, frozen orlyophilized. The internal environment of ISCOMs is different from thesurrounding liquid phase, as small water-soluble molecules cannot beretained [1]. ISCOMs are soluble in water and can as such be used tocarry hydrophobic molecules (e.g. antigens) in aqueous solutions. One ofthe biological properties of ISCOMs is their ability to penetrate cellmembranes allowing embedded molecules to access the interior of cells.This ability is probably due to their hydrophobic structure and theircontent of Quil A, a saponin that complexes cholesterol membranes. QuilA is as such surface-active and hemolytic and may have adverse effectswhen applied in vivo. Upon formulation of Quil A into ISCOMs thehemolytic property is partly inhibited lowering this undesired effect.The fate of ISCOMs in cells has not been reveled in details, although asubstantial part of ISCOM-bome antigen has been demonstrated in thecytosol of both phagocytic and non-phagocytic antigen-presenting cells(e.g. macrophages, dendritic cells and B-cells) [2]. Other reportsdescribe the finding intact and partially-degraded ISCOMs within thephagosomes of macrophages in close association with the pahgosomalmembranes [3].

Many proteins (antigens) can be inserted into the ISCOM-structure bymixing of Quil A, cholesterol and phospholipid (phosphatidylcholine)with the protein of interest. During the formation of the ISCOMs proteinis entrapped and are stably embedded. This approach is often efficientfor amphipathic proteins like membrane-spanning glycoproteins, andhydrophilic proteins chemically modified by the coupling of fatty acids(e.g. palmitification). However, some hydrophilic proteins like bovineserum albumin and ovalbumin reveal hydrophobic regions by treatment atlow pH enabling insertion without chemical modification [4; 5]. Othersubstances can be linked to the preformed structures (ISCOMs orISCOM-matrix) using bifunctional reagents. Substitutingphosphaditylcholine with phosphaditylethanolamine during the formationof ISCOMs facilitate chemical modification by supplying reactive aminogroups. However both the incorporation method and the chemical linkingmethod requires the compounds to be soluble in water:detergent. Thisrequirement limits the variety of substances embeddable. Compounds thatdo not directly dissolve in aqueous solutions may show soluble inmixtures of organic solvent, water and detergent. In this report weinvestigate if ISCOMs can be formed in mixtures of water and differentpolar organic solvents and at what concentrations. The three solvents ofinterest are N,N′-dimethylsulfoxide (DMSO), dimethylformamide (DMF) andethanol (EtOH), all of which are mixable with water at allconcentrations. Also, we address if the use of these solvents render newcompounds possible for insertion into ISCOMs. The examples are asynthetic lipopeptide and the fluorescent lipophilic tracer Dil.

It is generally assumed that ISCOMs prepared by the use of a detergentwith high CMC (critical micelle concentration) can be purified bydialysis. The dialysis step removes low molecular weight components notincorporated into the ISCOM-structures. However, it has not beeninvestigated if the detergent forms part of the ISCOMs rendering removalby dialysis impossible. All ISCOMs described here are prepared by theuse of Mega 10 (N-Decanoyl-N-methylglucamide). To investigate if Mega 10is completely removed during dialysis, radiolabeled Mega 10 wassynthesized in order to trace the detergent in ISCOMs.

Materials and Methods

Synthesis of Mega 10 and ¹⁴C-Mega 10.

In order to verify the synthesis path 20 mmol of decanoic acid (3.44 g)were dissolved in 20 ml of ice-cold ether and 2 g of pyridine (2.04 ml).After 10 min a slight excess of ethylchloroformate (22 mmol; 2.39 g=2.1ml) was added while vigorously stiffing. The precipitate was allowed toform for 10 min and was removed by filtration. The filtrate was added toa solution of N-methyl-Dglucamine (2 mmol; 3.9 g) in warm methanol(64.5° C.) and was allowed to react for 1 hour at room temperature.Then, the solution was stored overnight at 4° C.

After the removal of ether and methanol by evaporation the remaining oilwas dissolved in 80 ml of ether and 7 ml of methanol and heated to 40°C. The mixture was allowed to cool slowly upon which a white precipitateformed and the precipitation was continued overnight at 4° C. whilestirring. White crystalline Mega 10 was collected by filtration anddried; total yield 5.0 g (74%).

The quality of the product was compared with Mega 10 obtained fromSigma-Aldrich by reversephase HPLC; no differences in HPLC-profiles wereobserved (data not shown).

In the case of ¹⁴C-Mega 10 the same procedure was followed, although 1mg of ¹⁴C-decanoic acid (sodium salt) was added to the solution ofunlabeled decanoic acid in ether. ¹⁴C-decanolc acid was obtainedSigma-Aldrich, St. Louis, Mo. The total yield of ¹²C/¹⁴C-Mega 10 was 4.0g (59%).

Preparation of Iscoms.

Mega 10 (N-Decanoyl-N-methylglucamide) was purchased from Sigma-Aldrich,St. Louis, Mo. and used as a 20% stock solution. Dil(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) wasobtained from Molecular Probes, Eugene, Oreg. and dissolved in DMF orEtOH before use (1.0 mg/ml). Cholesterole and phophatidylcholine(Epikuron 200S, Lucas Meyer Gmbh, Germany) was dissolved in 20% Mega 10at a concentration of 1% w/v with respect to each component and storedat −20° C. Quil A was prepared as described by Dalsgaard [6] and storedin solution at −20° C. until used.

ISCOMs were prepared by combining Quil A, cholesterol andphophatidylcholine with the component to be inserted (e.g. lipopeptide).ISCOMs were allowed to form for 4 hours with stirring at 35° C. (roomtemperature for experiments with ¹²C/¹⁴C-Mega 10) followed by dialysisagainst phosphate buffered saline (pH 7.2) in Slide-A-Lyzer® cassettes(Pierce, Rockford, Ill.) or dialysis tubing (Visking, London, UK), MWcut-off 10,000. The concentration in the reaction mixture of Quil Aranged 0.9 mg/ml to 1.3 mg/ml for the different preparations (seeSection 3), whereas the concentration of cholesterol andphophatidylcholine was 0.03% w/v in all experiments. The finalconcentration of Mega 10 was 4% w/v unless otherwise stated.

Purification of ISCOMs.

Sucrose gradients were prepared from 25% (w/v) sucrose in phosphatebuffered saline (PBS) pH 7.2. Polyallomer™ centrifuge tubes (size 13×51mm, Beckmann Instruments) were filled with 4.5 ml sucrose solution andstored at −20° C. Upon usage the required number of tubes was allowed tothaw slowly at 4° C., typically overnight, whereby the gradient formed[7]. Gradients were allowed to equilibrate at room temperature for atleast 1 hour before ISCOM-preparations were applied in a volume of 0.5ml or less. Centrifugation was performed in a Beckmann Instruments rotortype SW55Ti at 20° C., 50,000 rpm for 3 hours. Intact ISCOMs werecollected through a diode array UV-detector (Perkin Elmer) and monitoredat 210 nm [8].

Amino Acid Analysis.

The Pico-Tag System® from Waters Corporation was used for determinationof peptide content of ISCOMs. Hydrolysis was preformed in a Pico-Tagworkstation at 150° C. for 1 hour using 6N HCl and phenol, followed byderivatization with phenylisothiocyanale (PITC) according to thePico-Tag protocol. In order to obtain quantitative measurementISCOM-structure was disrupted by treatment with 1:1 dichloromethane(DCM) and acetonitril (ACN) for 10 min at room temperature after whichsolvent was evaporated in vacuum (se discussion in Section 3.2).

Results and Discussion

Formation of ISCOM-Matrix in the Presence of Organic Solvent.

The general procedure for the formation of ISCOMs were as described byDalsgaard et al. [9], although the concentration of Quil A was aslightly higher (1.3 mg/ml; 0.13% w/v), Cholesterol, phosphatidylcholineand organic solvent (DMSO, DMF or EtOH) were combined and mixed beforeaddition of Quil A. The initial series of experiments were performed atroom temperature giving rise to precipitation of the detergent in anunpredictable manner. By equilibrating the reaction mixture at 35° C.before the addition of Quil A and during the formation of ISCOM-matrixprecipitations were avoided. Also, it showed necessary to maintain anelevated temperature during the first hours of dialysis. Quil A, ISCOMsand lipids constitute a substrate for microbiological growth and it isgenerally advantageous to dialyze at temperatures below 5° C. In orderto avoid precipitation the dialysis buffer was kept at approx. 35° C.for the initial 34 hours then allowed to cool to room temperature.Dialysis was continued for at least 6 hours before the beaker was placedat 4° C. Dialysis was carried out in Slide-A-Lyzer® (MW cut-off 10,000)cassettes from Pierce, different dialysis systems may require differenttiming.

The influence of organic solvent on the formation of ISCOM-matrix wasevaluated by visual inspection by electron microscopy (EM). The primarycriterion was the shape and uniformity of the structures, secondary thenumber of ISCOM particles. The formation of ISCOM-matrix was unaffectedby the presence of DMSO up to a concentration of 20% v/v (FIG. 9A);above this limit the yield was affected. With 25% DMSO intact structurescould be observed but the number of ISCOMs were reduced. The samepattern was observed for DIMF. Here intact ISCOM-structures could beproduced at high concentrations with a DMF concentration at or below25%. The effect of DMF above 25% was a limited numbers of ISCOMparticles although intact with respect to appearance. EtOH wascompatible with the process at concentrations up to approx. 15%. Somediverging structures appeared among intact structures prepared at thisconcentration. Adding 20% of ethanol to the reaction mixture resulted ina very limited amount of intact ISCOM particles and clearly mis-shapenstructures (FIG. 9B). It can be speculated, that the concentration ofQuil A and lipids as well as the concentration of detergent (Mega 10)may bias the sensitivity of ISCOM-formation towards the concentration oforganic solvent. In these experiments the final Mega 10 concentrationwas 4% vtw. In order to transfer these findings it should be consideredto utilize equivalent concentrations of detergent. As a conclusion, theaddition of solvent should be limited to:

Maximum concentration (v/v) DMSO 20% DMF 25% EtOH 15%

Embedding of Lipopeotide into ISCOMs.

In order to incorporate lipopeptides into ISCOMs the lipopeptide must bebrought into solution. The solubility of lipopeptides depends both uponthe actual peptide sequence and the lipid tail. The lipopeptideNP₁47-S-Pam employed here consists of peptide sequence of 12 amino acidslinked to palmitic acid by a thioester bond. The lyophilized lipopeptideis practically insoluble in water:detergent but readily dissolves inDMSO. However, solutions in DMSO can be further diluted with waterdetergent making lipopeptide availably for embedding into ISCOMs. In thecase of NP₁47-S-Pam a final concentration of approx. 0.5 mg/mllipopeptide in 4.5% Mega 10, 8% v/v DMSO was used for the formation ofISCOMs. We found, that stepwise dilution with 10% Mega 10 followed bystepwise addition of water to obtain the desired concentration oflipopeptide worked consistently without precipitation of lipopeptide.Both detergent as well as water was added in three rounds withintermediate mixing. By addition of cholesterol, phosphatidylcholine andQuil A (0.13% w/v) ISCOMs formed spontaneously.

Sucrose-gradient centrifugation was used to purify intact ISCOMs.Gradients were scanned through a diode-array UV-detector to measurepeptide contents and ISCOMs were collected as previously described [8].ISCOMs are readily detected at 210 nm whereas peptides containingaromatic residues have a distinct absorption at 280 nm. The absorptionof empty ISCOMs at this wavelength is very limited and theA₂10/A₂80-ration related to the peptide contents. However, one mustemploy different methods in order to determine the peptide contentsexactly. General protein assays like the bicinchoninic acid (BCA) methodis often used to determine polypeptide contents of ISCOMs. Such assaysare often sensitive to the amino acid composition requiring solutions ofthe same peptide to be used as reference instead of proteinconcentration standards.

An alternative method is standard amino acid analysis like the Pico-TagSystem® from Waters Corporation base on acidic hydrolysis,derivatization by phenylsothiocyanate (PITC) and reverse-phaseHPLC-chromatography. Total peptide contents can be calculated from eachamino acid and the anticipated relative amino acid composition verified.We find lyophilized ISCOMs to be compatible with the Pico-Tag protocolalthough ISCOM-structure must be disrupted prior to analysis bydichloromethane (DCM) and acetonitril (ACN). Undisrupted ISCOMs protectsembedded peptides from hydrolysis during the harsh conditions of theacidic hydrolysis during the amino acid analysis (FIG. 10), emphasizingthe high stability of the ISCOM structure and the difference in chemicalenvironment within ISCOMs.

Lovgren et al. have previous proposed immunization with lipid-tailedpeptides inserted into ISCOMs [10]. In this study an amide-linkedlipopeptide originating from foot-and-mouth disease virus (VP1 144-159)was embedded into ISCOMs after dissolving lipopeptide in DMSO. ISCOMswere used for immunization, but showed inefficient in inducingantibodies (personal communication) indicating the embedding approach tobe inapplicable for peptide vaccines. Consequently other methods havebeen investigated including the use of preformed ISCOMs as peptidecarriers. Immunization with a 17-mer porcine growth hormone peptideconjugated to influenza virus ISCOMs by the bi-functional coupling agentmaleimidohexanoyl-N-hydroxy-succinimide ester showed efficient [11],establishing ISCOMs as an alternative to the protein carriers likekeyhole limpet haemocyanin (KLH). However, in resent experiments withthioester-linked lipopeptide including mouse CTL-epitopes derived frominfluenza virus (like NP₁47-S-Pam) and a protective epitope from CanineParvo Virus (CPV) lipopeptide embedded into ISCOMs were efficient inpriming immunological response [12; 13]. These findings may shownimportant for future combinations of peptides and ISCOMs and do supportthe theory stated by Beekman et al. that the low immunogenicity ofamide-linked lipopeptides in ISCOMs is due to the high chemicalstability of the amide bond rather the embedment into the structure ofISCOMs itself [14].

Preparation of Dyed Iscoms.

The use of organic dissolvent was advantageous when incorporatingsparingly water-soluble lipopeptides into ISCOMs. In order toinvestigate if this method was applicable for other compounds thanlipopeptides, ISCOMs containing the long-chain dialkylcarbocyanine dyeDil was prepared. Labeling of pre-formed ISCOMs and ISCOM-matrix withDil has previously been described by Claassen et al. [15] by adaptationof methods developed for labeling of liposomal membranes [16]. Dil wasadded to ISCOMs at a concentration of 160 μg/ml (equivalent to 200 μg/mgISCOM) whereby up to 80% of the label was incorporated into theISCOM-structures while unincorporated dye did precipitate due to lowsolubility in the aqueous buffer.

In the present study Dil was embedded into ISCOMs by adding Dildissolved in DMF or EtOH to cholesterol and phophatidylcholine in Mega10 followed by addition of Quil A. The concentration of Dil was 150μg/ml giving rise to a DMF/EtOH concentration of 15% v/v in the reactionmixture.

In order to prevent precipitation of the dye the concentration of Mega10 was adjusted to 5% w/v before the addition of Dil. After addition ofDil and Quil A (1.3 mg/ml) the concentration of detergent was 4%.

Independently of organic solvent used (DMF or EtOH) no precipitations ofdye were observed during the formation reaction or after dialysisindicating quantitative incorporation of Dil. This was confirmed bydensity centrifugation on sucrose-gradients showing a distinct yellowband of ISCOMs with no staining outside of the ISCOM fraction (data notshown). Maximum absorption of Dil inserted into ISCOMs is observed at552 nm [17] and the amount of dye incorporated was compared to astandard solution of Dil in methanol using a Shimadzu UV-150-02spectrophotometer:

Percent incorporation Dil in DMF 93/104 Dil in EtOH 97/85 

Analysis of the Content of Detergent in ISCOMs

In the first experiment the kinetics of the Mega 10-dialysis wasinvestigated. ISCOM-matrix was formed in the presence of 7% w/v12C/14C-Mega 10 and Quil A at a concentration of 0.9 mg/ml. From a totalvolume of 18.3 ml sixteen aliquots of 1 ml were taken and set up fordialysis against water in 8 beakers of 1000 ml (2×1 ml bags per 1000 mlwater). Four beakers were placed at room temperature and four at 4° C.with stirring. On each of the following days one beaker from bothtemperatures were taken and the contents of the individual dialysis bags(2×2 bags) counted after addition of water in order to compensate fordifferences in volume. Table A lists the amount of ¹⁴C-Mega 10 remainingin each bag after dialysis for a certain period of time given as thepercentage of cpm (counts per minute) relative to the initial¹⁴C-contents. The results show that dialysis at room temperature isapproximately one day ahead of dialysis at 4° C. However, the sameend-point concentration of ¹⁴C-Mega 10 was reached within four days(0.4-1%) despite of temperature. Anticipating that Mega 10 diffusesfreely across the dialysis membrane one would expect the contents ofdialysis bags to represent 0.51% of the total ¹⁴C-Mega 10, as the volumeof two bags (from one beaker) at equilibrium was 5.1 ml leaving approx.995 ml in the beaker (5.1 ml/995 ml*100%=0.51%). And it is concludedthat although dialysis at room temperature is faster than dialysis at 4°C. both methods are equally efficient in removing Mega 10 fromISCOM-matrix.

TABLE A Equilibrium-dialysis of ¹⁴C-Mega 10. Amount of detergentremaining in dialysis bags at different time points given as percentageof initial cpm. Dialysis buffer (water) was not replaced. Time ofdialysis 1 day 2 days 3 days 4 days Room 2.1 0.9 0.9 0.4 temperature 2.10.9 0.9 0.7 4° C. 14.5 2.2 1.0 0.7 13.5 2.3 1.3 1.1

In order to further investigate if Mega 10 could be completely removedfrom ISCOM-matrix a second experiment was designed. Here, four dialysisbags containing 1 ml of ISCOM-matrix prepared with 3.3% w/v ¹²C/¹⁴C-Mega10 was placed in individual beakers containing 1000 ml of water.Dialysis was performed at room temperature with stirring. Each day onebeaker was taken and the contents of the dialysis bag counted afterwhich the dialysis buffer (water) of the remaining beakers was replaced.The measured contents of ¹⁴C-Mega 10 relative to the concentrationbefore dialysis was initiated are given in Table B.

It is concluded, that only two days of dialysis with one replacement ofthe water is required in order to obtain equilibrium. This issignificantly faster than in the previous experiment where equilibriumwas reached within 3-4 days. Furthermore, the efficiency by which Mega10 was removed increased by a factor of approx. 10, as only 0.07% of theoriginal amount of detergent remains within the dialysis bag. Thisfinding indicates that only a negligible amount of detergent remainsassociated with the ISCOM-structure after dialysis.

TABLE B End-point-dialysis of ¹⁴C-Mega 10. Amount of detergent remainingin bags after daily replacement of dialysis buffer (water). Time ofdialysis 1 day 2 days 3 days 4 days Room 0.23 0.07 0.06 0.07 temperature

REFERENCES

-   [1] Kersten, G. F., Spiekstra, A., Beuvery, E. C., &    Crommelin, D. J. (1991) On the structure of immune-stimulating    saponin-lipid complexes (iscoms). Biochim. Biophys. Acta, 1062,    165-171.-   [2] Villacres, M. C., Behboudi, S., Nikkila, T., Lovgren-Bengtsson,    K., & Morein, B. (1998) Internalization of iscom-bome antigens and    presentation under MHC class I or class II restriction. Cell    Immunol., 185, 30-38.-   [3] Watson, D. L., Lovgren, K., Watson, N. A., Fossum, C., Morein,    B., & Hoglund, S. (1989) Inflammatory response and antigen    localization following immunization with influenza virus ISCOMs.    Inflammation, 13, 641-649.-   [4] Morein, B., Ekstrom, J., & Lovgren, K. (1990) Increased    immunogenicity of a non-amphipathic protein (BSA) after inclusion    into iscoms. J. Immunol. Methods, 128, 177-181.-   [5] Heeg, K., Kuon, W., & Wagner, H. (1991) Vaccination of class I    major histocompatibility complex (MHC)-restricted murine CD8+    cytotoxic T lymphocytes towards soluble antigens:    immunostimulating-ovalbumin complexes enter the class I    MHC-restricted antigen pathway and allow sensitization against the    immunodominant peptide. Eur. J. Immunol., 21, 1523-1527.-   [6] Dalsgaard, K. (1974) Saponin adjuvants. 3. Isolation of a    substance from Quillaja saponaria Molina with adjuvant activity in    food-and-mouth disease vaccines. Arch. Gesamte Virusforsch., 44,    243-254.-   [7] Chanas, A. C. & Johnson, B. K. (1980) Sucrose density gradient    formation by freezing and thawing. Med. Lab Sci., 37, 89-90.-   [8] Beekman, N., Kamstrup, S., & Dalsgaard, K. (1995) Method for    monitoring ultracentrifuge gradients using a high-performance liquid    chromatographic diode array detector. Anal. Biochem., 228, 168-169.-   [9] Stewart-Tull, D. E., Dalsgaard, K., Lovgren, K., &    Lindblad, E. B. (1995) The Theory and Practical Application of    Addjuvants. John Wiley & Sons Ltd.-   [10] Lovgren, K. & Morein, B. (1988) The requirement of lipids for    the formation of immunostimulating complexes (iscoms). Biotechnol.    Appl. Biochem., 10, 161-172.-   [11] Lovgren, K. & Larsson, M. (1994) Conjugation of synthetic    peptides to earlier iscoms: factors affecting the immuhogenicity of    the conjugate. J. Immunol. Methods, 173, 237-243.-   [12] Kirkby, N., Schaaper, W. M., Fomsgaard, A., &    Dalsgaard, K. (2000) Vaccination by lipopeptides. Synthetic T-cell    epitopes palmitoylated by thioester linkage induce CTLs in vivo by    subcutaneous injection. Submitted for publication.-   [13] Kirkby, N. & Dalsgaard, K. (2000) Comparison of different    adjuvant systems for immunization with Canine Parvovirus. Submitted    for publication.-   [14] Beekman, N. J., Schaaper, W. M., Turkstra, J. A., &    Meloen, R. H. (1999) Highly immunogenic and fully synthetic    peptide-carrier constructs targetting GnRH. Vaccine, 17, 2043-2050.-   [15] Claassen, I., Osterhaus, A., Boersma, W., Schellekens, M. &    Claassen, E. (1995) Fluorescent labelling of virus, bacteria and    iscoms: in vivo systemic and mucosal localisation patterns. Adv.    Exp. Med. Biol., 371B, 1485-1489.-   [16] Claassen, E. (1992) Post-formation fluorescent labelling of    liposomal membranes. In vivo detection, localisation and    kinetics. J. Immunol. Methods, 147, 231-240.-   [17] Claassen, I. J., Osterhaus, A. D., & Claassen, E. (1995)    Antigen detection in vivo after immunization with different    presentation forms of rabies virus antigen: involvement of marginal    metallophilic macrophages in the uptake of immune-stimulating    complexes. Eur. J. Immunol. 25, 1446-1452.

Example 2 Preparation of DNA-Binding Complexes According to theInvention

This example demonstrates how to prepare micro-particles with thecapability of forming an association with DNA by means of anelectrostatic interaction The complexes were prepared by substituting afraction of cholesterol for DC-cholesterol during the formation of thecomplexes.

Stock solution of sterol and phospholipid. Three lipid stock solutionswas prepared by dissolving Cholesterol,3β-[N-(N′,N′-Dimet-hylaminoethane)-Carbamoyl] Cholesterol(DC-Cholesterol, Avanti Polar Lipids) and phophatidylcholine (Epikuron200S, Lucas Meyer Gmbh, Germany) in 20% w/v Mega 10(N-Decanoyl-N-methyl-glucamide, Sigma-Aldrich). The concentration of thethree stocks were (w/v):

DC-Cholesterol Cholesterol Phosphaditylcholine Stock A 0.5% 0.5% 1.0%Stock B 0.25% 0.75% 1.0% Stock C 0.13% 0.87% 1.0%

Lipids were dissolved by agitation and heating to 40° C. Stock solutionswere stored at −20° C. until used.

Complex formation. A reaction mixture containing Quil A (Quillajasaponin) at a concentration of 1.7 mg/ml and 0.034% w/vphophatidylcholine in 5% w/v Mega 10 was prepared by diluting lipidstock A, B or C with 10% w/v Mega 10 and water. The final concentrationof DC-cholesterol and cholesterol varied due to the composition of theselected lipid stock solution:

Final conc. DC-Cholesterol Cholesterol Phosphaditylcholine Stock A0.017% 0.017% 1.0% Stock B 0.0085% 0.026% 1.0% Stock C 0.0043% 0.030%1.0%

Complex formation was initiated by the addition of Quil A and thereaction mixtures were incubated with magnetic stirring for 2 hours at30° C. by which the microparticles form.

Complex purification. Following incubation the micro-particlesuspensions were dialyzed against phosphate buffered saline (PBS) usingdialysis tubing with a molecular weight cut-off at 10,000 (Pierce,Slide-A-Lyzer, MWCO 10,000). Dialysis is continued for 24 hours at roomtemperature, with one replacement of dialysis buffer after 12 hours.

Evaluation of micro-particle structure. After dialysis ISCOMs werepassively absorbed on electron microscopy grids, dried and negativelystained with uranylacetate. All preparations yielded particles withcharacteristic cage-like structures know to ISCOMs.

Example 3 Degradation of Linear Plasmid DNA Bound to DNA-Binding ISCOMs

The aim of the present example is to examine whether DNA associated withDNA-binding ISCOMs are protected from degradation by DNase I. Lineraizedplasmid DNA was enzymatically labeled with ³²P. Labeled DNA was allowedto bind to ISCOMs and treated with or without DNAse I. ISCOMs werecollected by filtration and the amount of label retained was used as ameasurement of the amount of DNA protected from degradation.

Labeling of DNA. Plasmid pUC18 digested with BamH1 (Amersham-Pharmacia,pUC18 BamH1/BAP) was labeled using DNA polymerase I Kienow Fragment byadding 10 μCi [α-³²P]dGTP (Amersham-Pharmacia, AA0066) to 1 μg of DNA in25 μl EcoPol Buffer (New England BioLabs, 10 mM Tris-HCl, 5 mM MgCl₂, 5mM DTT, pH 7.5). By further addition of 1 unit Kienow Fragment (NewEngland BioLabs, M0212S) the reaction was initiated. After incubationfor 15 minutes at room temperature the reaction was stopped by heatingfor 5 minutes at 70° C. The volume was adjusted to 50 μl with water andunincorporated nucleotides was removed by spin-column purification usingMicroSpin S-400 HR Columns (Amersham-Pharmacia, 27-5140-01) as describedin the instructions manual.

After removal of unincorporated ³²P the plasmid was further diluted to400 μA by Eco-Pol Buffer (New England BioLabs) and the incorporationchecked by Cerenkov counting using a Packard Tri-Card 4000 scintillationcounter.

Binding of labeled DNA to ISCOMs. ISCOMs containingDC-cholesterol/cholesterol at a ration of 1:1 was allowed to associatewith labeled DNA. In two separate tubes (A. B) 100 μl of labeled linearplasmid (corresponding to approx. 150 ng) was mixed with 10 μg of ISCOMs(30 μl of ISCOMs phosphate buffered saline, pH 7.2) and incubated for 1h at room temperature. As a control experiment ordinary ISCOMscontaining no DC-cholesterol (10 μg in phosphate buffered saline) wasmixed with an equal amount of labeled DNA and the volume adjusted to 130μl. This sample (C) was incubated in parallel to the samples containingDC-cholesterol ISCOMs.

Degradation of DNA. After incubation of labeled DNA with ISCOMs, 10units DNase I (Stratagene, 600031) was added to tubes A (DNA-bindingISCOMs) and C (control ISCOMs). Before addition enzyme was diluted10-fold to 10 units/μl in DNase reaction buffer (40 mM Tris-HCl, 6 mMMgCl₂, 2 mM CaCl₂, pH 7.5). DNase was allowed to digest DNA for 1 hourat 37° C., after which the reaction was stopped by addition of 5 μl 500mM EDTA pH 8.0.

Separation of degraded DNA and ISCOM-bound DNA. Both the DNase treatedsamples (A, C) and the non-digested sample (B) was transferred tomicro-centrifuge filters (Millipore, Ultrafree-MC, 30,000 NMWL FilterUnits) and forced through the filter membrane by centrifugation for 10minutes at 4.000×g. The effluent was discarded while the filter-cupswere transferred to scintillation-vials. The amount of label retained inthe filters was measured by adding 2 ml of scintillation fluid (Packard,Opti-Flour O) to each filter followed by counting on ³²P-programme inPackard Tri-Carb 4000 scintillation counter.

Results. It is anticipated that a linear correlation between themeasured amount of counts per minute (cpm) and the amount of intact DNAentrapped by the filters exists. The specific activity measured for thecontrol reaction (C) was 1525.+−0.305 cpm which, when compared to the18,235.+−0.456 cpm of the non-digested DNA in combination withDC-cholesterol (B) verifies the capacity of these ISCOMs to bind DNA.

Furthermore, when DNase was used to degrade DNA prior to filtration theactivity measured on the filter (A) was reduced to 7,315.+−0.183 cpm.This is lower than the un-digested sample (B) indicating that some ofthe label was released from the ISCOMs due to degradation of DNA byDNase I.

However, as the measurement of the digested sample (A) is at least afactor of 4 higher than the control (C) a substantial amount of the DNAis retained on the filters despite the DNase treatment. It is concludedthat the ISCOMs containing DC-cholesterol not only binds DNA but alsoprotects DNA, when bound, from degradation by DNase I.

Example 4 Determination of Zeta-Potentials of DNA-Binding ISCOMs

The parameter of zeta potential is a measure of the magnitude of therepulsion or attraction between particles. Its measurement relates tosome extent to the overall charge of particles but also to the stabilityof particles in dispersion. The surface charge of particles in polarliquids dose not directly correlate to the electrical potential at thesurface of the particle but to the potential that in the close vicinityof the particle.

The Zeta-potential of three different compositions of ISCOMs wereanalyzed using Zetasizer 1000 HS from Malvern. Instruments Ltd, UK.

Preparation of ISCOMs. Following the procedure in Example no. 2, ISCOMswere prepared as 25% or 50% of the cholesterol were substituted byDC-cholesterol during the formation of the particles. Also ISCOMs inwhich no DC-cholesterol were prepared by the analogous protocol. ISCOMswere purified by dialysis against phosphate buffered saline (0.85% NaCl,0.01M phosphate buffer, pH 7.2) and stored at −20° C. until used.

Measurement of Zeta-potential Prior to measurement ISCOMs were dilutedto a final concentration of 5-20 μg/ml with phosphate buffered saline(as above). For each measurement approx. 5 ml of diluted ISCOMs wereinjected into the measuring chamber.

Zeta-potentials were measured using standard procedures as described inthe instruments manual. For each injected sample the Zeta potential weremeasured five consecutive times (5×1 minute).

FIG. 11 illustrates the measurement of ISCOMs containing noDC-cholesterol. Each curve represents one of five measurements. Theaverage Zeta-potential was found to be approx. −50 mV, which issignificantly different from the data obtained from measurements ofISCOMs containing DC-cholesterol (DC-cholesterol to cholesterol ratio1:1 or 1:2) as illustrated in FIG. 8.

FIG. 12 illustrates the measurement of modified ISCOMs. DC-cholesterolto cholesterol ration was 1:1 (50% substitution). Similar to theexperiments described above and in FIG. 7, the Zeta-potential wasmeasured five times. The Zeta-potential was close to 0 mV with somevariation between measurements. When ISCOMs with a DC-cholesterol tocholesterol of 1:2 (25%) were measured, an average Zeta-potential ofapprox. −25 mV was observed (data not shown).

Example 5 Transfection with Plasmid DNA in Combination with DNA-BindingISCOMs

To investigate if the hemolytic property of ISCOMs could increase theuptake of DNA in cultured cells a reporter plasmid expressing greenflourescent protein (GFP) was transfected after binding toDC-cholesterol containing ISCOMs.

Preparation of DNA-ISCOM complexes. Plasmid DNA (2 μg) was combined with2 μg of ISCOMs containing DC-cholesterol and cholesterol at a ratio of1:1, as prepared in Example 2. This was done in a volume of 40 μl (10 mMTris-HCl, pH 7.5) and complexes were allowed to form for 1 h at roomtemperature. The volume was adjusted to 100 μl.

Preparation of DNA-Lipofectin complexes. To compare the efficiency ofISCOMS versus a well-know and efficient transfection reagent(Lipofectamin Reagent, Gibgo BRL, 18292-011) was included in theexperiment. Again, 2 μg of plasmid DNA was combined diluted to 50 μlwith water and combined with a solution of 50 μl (15 μl LipofectinReagent diluted with 35 μl water).

Transfection of cells. RDM4 cells were cultured in RPMI 1640 mediumsupplemented with 10% fetal calf serum (FSC). Cells (10⁵) were grown insmall tissues culture dishes (ø 40 mm) for 24-36 h prior totransfection. Before transfection cells were washed three times withserum-free medium. The DNA suspensions were added to 2 ml of serum-freemedium just prior to transfection and added to the cells. Cells wereincubated for 4 hours and then washed with medium containing serum.After removal of the transfection mixture cells were allowed to grow for24 h before the GFP-activity were tested.

Result of transfection. Fluorescent cells were counted in astero-microscope equipped with a ultraviolet light source and thefraction of transfected cells estimated. Transfection with Lipofectinwere highly efficient, as more than 80% of the cells were clearlyfluorescent. ISCOMs, where also capable to transfect cells, but withlower efficiency. Here, approx. 25% of the cells had the fluorescentphenotype although the intensity of the fluorescent light was quietvarying from cell to cell.

1. A particle structure comprising: (i) a sterol comprising a gonaneskeleton structure, wherein the sterol is anionic or neutral; (ii) asaponin isolated from a plant species selected from the group consistingof Quillaja saponaria, Saponaria officinalis, Gypsophila struthium,Aesculus hippocastanum, Silene jenisseenis, Acanthophyllum squarrosum,and combinations thereof, wherein said sterol and said saponin interactwith each other; and (iii) a cationic sterol comprising a gonaneskeleton structure and at least one moiety carrying a positive charge atpH 7.0.
 2. The particle structure of claim 1, wherein said saponincomprises the following formula:

wherein R₁ is —C—O—R₄, wherein R₄ is a linear or branched saccharidecomprising at least one monosaccharide moiety selected from the groupconsisting of D-glucose, D-galactose, D-glucuronic acid, D-galacturonicacid, L-rhamnose, L-arabinose, D-xylose, D-fucose, D-apiose, D-ribose,D-allose, D-quinovose, and combinations thereof; and wherein R₂ is—COOR₅, —C═O, or —C—OH, wherein R₅ is —H or a linear or branchedsaccharide, said saccharide comprising at least one monosaccharidemoiety selected from the group consisting of D-glucose, D-galactose,D-glucuronic acid, D-galacturonic acid, L-rhamnose, L-arabinose,D-xylose, D-fucose, D-apiose, D-ribose, D-allose, D-quinovose, andcombinations thereof; and wherein R₃ at each of positions C1, C2, C11,C15, C16, C21, C22, C23, C24, C26, C27, C29, and C30 is, independent ofthe other positions, —H, —OH; ═O; —COOH; —COOMe; or ══.
 3. The particlestructure of claim 2, wherein R₅ is a linear or branched saccharide,said saccharide comprising at least one monosaccharide, wherein said atleast one monosaccharide is substituted with acyl moieties selected froma group consisting of 3,5-dihydroxy-6-methyloctanoic acid,3,5-dihydroxy-6-methyloctanoic acid,5-O-α-L-rhamno-pyranosyl-(1→2)-α-L-arabino-furanoside, and5-O-α-L-arabino-furanoside.
 4. The particle structure of claim 2,wherein R₁ is a branched trisaccharide.
 5. The particle structure ofclaim 2, wherein R₃ of said saponin is —OH at C16, ═O at C23, and —H atpositions C1, C2, C11, C15, C21, C22, C24, C26, C27, C29, and C30. 6.The particle structure of claim 5, wherein R₅ is a linear or branchedsaccharide, said saccharide comprising at least one monosaccharide,wherein said at least one monosaccharide is substituted with acylmoieties selected from a group consisting of3,5-dihydroxy-6-methyloctanoic acid, 3,5-dihydroxy6-methyloc-tanoicacid, 5-O-α-L-rhamno-pyranosyl-(1→2)-α-L-arabino-furanoside, and5-O-α-L-arabino-furanoside.
 7. The particle structure of claim 6,wherein R₅ is a branched saccharide, said saccharide comprising sevenmonosaccharides, wherein said at least one monosaccharide is substitutedwith acyl moieties selected from a group consisting of3,5-dihydroxy-6-methyloctanoic acid, 3,5-dihydroxy-6-methyloc-tanoicacid, 5-O-α-L-rhamno-pyranosyl-(1→2)-α-L-arabino-furanoside, and5-O-α-L-arabino-furanoside.
 8. The particle structure of claim 7,wherein the saponin is QuilA.
 9. The particle structure of claim 8,wherein the saponin is substantially pure.
 10. The particle structure ofclaim 1, wherein the saponin is isolated from Quillaja saponaria. 11.The particle structure of claim 10, wherein the saponin is selected fromthe group consisting of QA1, QA2, QA3, QA4, QA5, QA6, QA7, QA8, QA9,QA10, QA11, QA12, QA13, QA14, QA15, QA16, QA17, QA18, QA19, QA20, QA21,QA22, and combinations thereof.
 12. The particle structure of claim 1,wherein said sterol of (i) is selected from the group consisting ofcholesterol, lanosterol, lumisterol, stigmasterol, sitosterol,ergosterol, thiocholesterol, nordihydro-epi-andosterol, and combinationsthereof.
 13. The particle structure of claim 1, wherein said cationicsterol of (iii) comprises the following formula:

wherein X is —OOC(CH₂)₃N⁺(CH₃)₃, —OOC(CH₂)₂COO(CH₂)₂N⁺(CH₃)₃,—OCONH(CH₂)₂N(CH₃)₂; —OCONH(CH₂)₂N⁺(CH₃)₃Cl⁻;


14. The particle structure of claim 13, wherein X is—OCONH(CH₂)₂N(CH₃)₂.
 15. The particle structure of claim 13, wherein Xis —OCONH(CH₂)₂N⁺(CH₃)₃Cl⁻.
 16. The particle structure of claim 1,further comprising a bioactive agent.
 17. The particle structure ofclaim 16, wherein the bioactive agent is selected from the groupconsisting of DNA, RNA, a contrast agent, an immunogenic determinant, anantigenic determinant, a chemotherapeutic agent, a peptide, a nucleicacid of either natural or synthetic origin, and combinations thereof.18. The particle structure of claim 17, wherein the bioactive agent isan immunogenic determinant.
 19. The particle structure of claim 1further comprising a targeting ligand selected from the group consistingof proteins, antibodies, antibody fragments, hormones, hormoneanalogues, glycoproteins, lectins, peptides, polypeptides, amino acids,sugars, monosaccharides, polysaccharides, carbohydrates, vitamins,steroids, steroid analogs, cofactors, nucleosides, nucleotides,nucleotide acid constructs, polynucleotides, optionally constructs orpolynucleotides of either natural or synthetic origin, and combinationsthereof.
 20. The particle structure of claim 19, wherein the targetingligand is a carbohydrate.
 21. The particle structure of claim 1, furthercomprising a compound selected from the group consisting ofphosphatidylcholine, phosphatidylethanolamine, triglycerides, fattyacids, and hydrophobic amino acid residues, and combinations thereof.22. The particle structure of claim 21, wherein said compound isphosphatidylcholine.
 23. The particle structure of claim 1, furthercomprising a compound selected from the group consisting of a quaternaryammonium compound, a dialkyldimethylammonium compound,dioctadecyldimethyl ammonium chloride, dioctadecyldimethyl ammoniumbromide, dioctadecyl/octadienyldimethyl ammonium chloride,dioctadecyl/octadienyld-imethyl ammonium bromide,dimethyldioctadecylammonium bromide (DDAB), dodecyltrimethylammoniumbromide, a hexadecyltrimethylammonium compound, mixedalkyltrimethylammonium bromide (Cetrimide per BP); atetradecyltrimethylammonium compound, and a combination of saidcompounds, wherein said compound is capable of associating with saidbioactive agent.
 24. The particle structure of claim 1, wherein a ratiobetween the sterol of (i) and the cationic sterol of (iii) is less than1000:1 to more than 1:4.
 25. The particle structure of claim 1,comprising: (i) cholesterol; (ii) QuilA; (iii) DC-Chol; (iv)phosphatidylcholine; and (v) a bioactive agent.
 26. The particlestructure of claim 1, comprising: (i) cholesterol; (ii) QuilA; (iii)DC-Chol; (iv) phosphatidylcholine; and (v) a targeting ligand.
 27. Theparticle structure of claim 1, wherein said particle has a sphericalshape and a diameter in the range of from 25 nm to 75 nm.
 28. Theparticle structure of claim 1, wherein said particle is in the form of acage-like matrix.
 29. The particle structure of claim 1, characterizedby a zeta potential of more positive than about −50 mV.
 30. The particlestructure of claim 29, characterized by the zeta potential of about −40mV to about −10 mV.
 31. A method of preparing a particle structure,comprising mixing together: (i) a sterol comprising a gonane skeletonstructure, wherein the sterol is anionic or neutral; with (ii) a saponinisolated from a plant species selected from the group consisting ofQuillaja saponaria, Saponaria officinalis, Gypsophila struthium,Aesculus hippocastanum, Silene jenisseenis, Acanthophyllum squarrosum,and combinations thereof; (iii) at least one organic solvent; and (iv) acationic sterol comprising a gonane skeleton structure and at least onemoiety carrying a positive charge at pH 7.0; wherein the ingredients i)to iv) may be mixed simultaneously, or sequentially, in any order. 32.The method of claim 31, further comprising the addition to the mixtureof one or more bioactive agents.
 33. The method of claim 31, furthercomprising addition to the mixture of a compound selected from the groupconsisting of phosphatidylcholine, phosphatidylethanolamine,triglycerides, fatty acids, hydrophobic amino acid residues, andcombinations thereof.
 34. The method of claim 31, further comprising:(v) removing surplus reactants and/or purifying the prepared complexes.35. The method of claim 31, wherein said sterol of (i) is selected fromthe group consisting of cholesterol, lanosterol, lumisterol,stigmasterol, sitosterol, ergosterol, thiocholesterol,nordihydro-epi-andosterol, and combinations thereof.
 36. The method of31, wherein the cationic sterol of (iii) comprises the followingformula:

wherein X is —OOC(CH₂)₃N⁺(CH₃)₃, or —OOC(CH₂)₂COO(CH₂)₂N⁺(CH₃)₃, or—OCONH(CH₂)₂N(CH₃)₂; or —OCONH(CH₂)₂N⁺(CH₃)₃Cl⁻; or


37. The method 31, wherein the solvent is present in a (vol/vol) amountof 25% (vol/vol), 10% (vol/vol), 4% (vol/vol), 1% (vol/vol), or 0.1%(vol/vol).
 38. A vaccine composition comprising the particle structureof claim 1.